Peptide PAC1 antagonists

ABSTRACT

Novel peptides that bind to human PAC1 are provided. These peptides that are antagonists of PAC1 are useful in a number of PAC1 related disorders, including the treatment and/or prevention of headache disorders, including migraine, such as acute migraine.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the national stage application under 35 U.S.C. § 371of International Application No. PCT/US2018/035597, having aninternational filing date of Jun. 1, 2018; which claims the benefitunder 35 U.S.C 119(e) of U.S. Provisional Application No. 62/514,440,filed Jun. 2, 2017, all of which are incorporated by reference herein intheir entireties.

REFERENCE TO THE SEQUENCE LISTING

The present application contains a Sequence Listing, which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The computer readable format copy of theSequence Listing, which was created on May 30, 2018, is namedA-2135-WO-PCT_Sequence_Listing_ST25.txt and is 271 KB bytes in size.

BACKGROUND

Migraine is characterized by headaches that can involve significantpain, and is often accompanied by nausea, vomiting, and extremesensitivity to light (photophobia) and sound (phonophobia), and issometimes preceded by sensory warning symptoms or signs (auras). It is ahighly prevalent disease worldwide with approximately 12% of theEuropean population, and 18% of women, 6% of men in the United Statessuffering from migraine attacks (Lipton et al, Neurology, Vol.68:343-349, 2007; Lipton et al., Headache, Vol. 41:646-657, 2001).Additionally, migraines are associated with a number of psychiatric andmedical comorbidities such as depression and vascular disorders (Buse etal., J. Neurol. Neurosurg. Psychiatry, Vol. 81:428-432, 2010; Bigal etal., Neurology, Vol. 72:1864-1871, 2009). There is a significant unmetneed for effective therapies for migraine. Most of the current migrainetherapies are either not well tolerated or ineffective (Loder et al.,Headache, Vol. 52:930-945, 2012; Lipton et al, 2001); thus, migraineremains an unmet medical need.

The exact mechanism of migraine pathophysiology has been debated sincethe 17th century but is still not fully clarified, even thoughscientists have made a lot of progress in understanding it. A majorcomponent of migraine pathogenesis involves the activation of thetrigeminovascular system. The release of trigeminal and parasympatheticneurotransmitters from perivascular nerve fibers (Sinchez-del-Rio etal., Curr. Opin. Neurol., Vol. 17(3):289-93, 2004) result invasodilation of the cranial blood vessels and has been suggested to beassociated with the onset of migraine headaches (Edvinsson, Cephalagia,Vol. 33(13): 1070-1072, 2013; Goadsby et al., New Engl J Med., Vol.364(4):257-270, 2002).

Pituitary adenylate cyclase-activating peptide (PACAP) and its receptors(PAC1, VPAC1 and VPAC2) are present in sensory neurons and in vascularsmooth muscle related to the trigeminovascular system, a key circuitryin migraine pain. Recent data point to an involvement of PACAP, inparticular the PAC1 receptor, in the pathophysiology of migraine(Rahmann A. et al. Cephalalgia 2008; 28:226-36). PAC1 receptor is aclass II G-protein coupled receptor that modulates adenyl cyclaseactivity and cAMP signaling. Vasoactive intestinal peptide and PACAPmediate their effect via VPAC1 and VPAC2 receptors with almost equalaffinity, whereas PACAP has much high affinity to the PAC1-receptor.Considering that PACAP-38 is a trigger of migraine attacks, thePAC1-receptor may therefore be a putative target for migraine treatment(Edvinsson L., Br J Pharmacol 2014). Furthermore, recent clinicalstudies have demonstrated that the intravenous administration of PACAPcan induce middle meningeal artery vasodilation and headache in bothmigraineurs and healthy subjects. Id.

Maxadilan is a PAC1 receptor agonist (Banki E. et al., Neuropharmacology2014; 85:538-4). A recent study showed that PAC1 receptor activation byMaxadilan could inhibit the acute neurogenic arterial vasodilation andthe plasma protein release from the venules. Considering the findingsthat demonstrated the migraine attack induction by PACAP, the PAC1receptor can represent a promising candidate as a therapeutic target(Rainero I. et al. J Headache Pain 2013; 14).

SUMMARY

In one aspect, the invention includes novel peptides that areantagonists of the PAC1 receptor for treatment of migraine.

The peptides of Formula I, or a pharmaceutically acceptable saltthereof, are contemplated by the invention.X⁰X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹X¹⁰X¹¹X¹²X¹³X¹⁴X¹⁵X¹⁶X¹⁷X¹⁸X¹⁹X²⁰X²¹X²²X²³X²⁴X²⁵X²⁶X²⁷X²⁸X²⁹X³⁰X³¹X³²X³³X³⁴X³⁵X³⁶X³⁷X³⁸X³⁹X⁴⁰X⁴¹X⁴²X⁴³X⁴⁴  (FormulaI)

All variations listed below are contemplated as independent from eachother.

In one aspect, X⁰ is Gly, Ser, Gly-Ser-, Ala, Met, [Pra], Gly-Met- or isabsent.

For example, X¹ can be Cys if X⁵ is Cys or X¹ can be Ser if X⁵ is Ser,Thr, or is absent. X² can be Asp, Glu, or an acidic amino acid residue,or can be absent. In another aspect, X³ is Ala, Pro, Ser, [Aib], or ahydrophobic amino acid residue, or is absent. In another example, X⁴ maybe chosen from Thr, Ser, Val, Tyr or a neutral, hyrdrophilic orhydrophobic amino acid residue, or may be absent. X⁵ can be Cys if X¹ isCys; or X⁵ is Ser if X¹ is Ser, or can be absent. Further, X⁶ can beLys, [Cit], His, Arg, or other basic amino acid residue or Gln, Asn,Phe, or other neutral amino acid residue, or Glu or other acidic aminoacid residue, or can be absent. In one aspect, X⁷ is Phe, Tyr, His or aneutral or hydrophobic amino acid residue, or is absent; X⁸ is Lys, Arg,or a basic amino acid residue, or is absent; X⁹ is Lys, Arg, or a basicamino acid residue, or is absent, and X¹⁰ is Ala, Cys, Gly, Leu, Gln,Val or a neutral amino acid residue, or is absent.

The invention provides PAC1 antagonistic peptides, wherein X¹¹ can beIle, Leu, Met, [Aib] or a hydrophobic amino acid residue, or can beabsent; X¹² can be Asp, Glu, or an acidic amino acid residue, or Ala,Val or other neutral amino acid residue or can be absent. Further, X¹³can be Asp, Glu, or an acidic or Ala, Val, [Aib] or other neutral aminoacid residue, or Lys, or is absent. In one aspect, X¹⁴ is Cys, Trp, Ile,Met, Ser or absent; X¹⁵ can be Gln, Ala, Phe, Ile, Leu, Met, Ser, Val,Trp, Tyr or other neutral amino acid, or Asp, [Cit], or a neutral oracidic amino acid residue, or His, Lys or other basic amino acidresidue, or absent; X¹⁶ can be Lys, Arg, His or other basic amino acidresidue or Phe, Ile, Leu, [Aib], Gln, Met, Thr, Val, [Pra], Trp, Tyr,Gly, His, Asn or other neutral or [hGlu] acidic amino acid residue, orabsent; X¹⁷ can be Ala, Gln, [Aib], Phe, Ile, Leu, Ser, Val, Trp, Tyr,Thr or other neutral amino acid residue, or [Cit], Lys, Arg, or otherbasic amino acid residue or Asp, or other acidic amino acid residue orabsent; X¹⁸ can be Ala, Leu, [Aib], [Pra], Ser or other neutral aminoacid residue, or Glu or other acidic amino acid residue, or Lys or otherbasic amino acid residue, or absent; X¹⁹ can be His, Lys, Arg or otherbasic amino acid residue or Ala, Phe, Ile, Leu, Met, Gln, Ser, Val, Trp,Tyr, [Aib], or other neutral or hydrophobic amino acid residue orabsent; X²⁰ can be Ala, [Aib], Tyr, or other neutral or hydrophobicamino acid residue, or His or other basic amino acid residue or absent.

The invention also provides that X²¹ can be Ala, Met, Ser, Val, [Aib],or a neutral amino acid residue, or absent; X²² can be Asn, His, Gln, ora basic or neutral amino acid residue, or Glu or other acidic amino acidresidue, or absent; X²³ can be Gly, Val, Thr, or a neutral amino acidresidue, or Glu or other acidic amino acid residue, or Lys or otherbasic amino acid residue, or absent; X²⁴ can be absent, or can be Pro,[Pip], [Sar], [Hyp], [DHP] or a neutral amino acid residue, or Arg orother basic amino acid residue; X²¹ can be absent, Ala, Gly, Tyr, [Pra],or a neutral amino acid residue; X²⁶ can be absent, Asn, Gln or aneutral amino acid residue, or -Leu-Gln-Thr-Ser-Val-; X²⁷ can be Ala,Gly, Ser, [Aib] or a neutral or hydrophilic amino acid residue, or Aspor other acidic amino acid residue, or is absent; X²⁸ can be absent,Val, Thr, Ala, or a neutral or hydrophilic amino acid residue, or Lys orother basic amino acid residue; X²⁹ can be absent, or can be Phe, Trp,Tyr, [Aib], or a neutral or hydrophobic amino acid residue; X³⁰ can beLys, [Aib], Ala, or [d-Ala] (also depicted as a) or a basic or neutralamino acid residue, or Glu or other acidic amino acid residue or absent.

In another aspect, X³¹ can be Glu, [Aib], Ala, Ser, Thr, Val, Leu or anacidic or neutral amino acid residue, Lys or other basic amino acidresidue or absent; X³² can be Cys, Leu, Ser, or is absent, or Arg; X³³can be Ala, Phe, Ile, Leu, Ser, Val, [SeMet], Met, [Nle], or ahydrophobic amino acid residue, or is absent: X³⁴ can be Lys, [Aib],Ala, Leu, Val or a basic or neutral amino acid residue, or Glu or otheracidic amino acid residue or absent; X³⁵ can be Gln, Phe, Asn, Thr,[Aib], Ala, Pro, Cys or neutral amino acid residue, or Glu or an acidicamino acid residue, or Arg or other basic amino acid residue or absent;X³⁶ can be Lys, [d-Lys] (also depicted as k), [Orn], Glu, Gly, Gln, His,Ala, [Aib], [NMeLys], Cys, Arg, Leu or a basic or neutral amino acidresidue, or absent; X³⁷ can be Lys, Ala, Gln, [Aib], Phe, Arg, or abasic or neutral amino acid residue, or absent; X³⁸ can be absent, Lys,Ala, [Aib], Glu, [Orn], Gln, Gly, [d-Lys](also depicted as k), [NMeLys],Phe, Leu, Asn, Arg, Thr, or an acidic or basic or neutral amino acidresidue; X³⁹ can be absent, or can be Glu, Ala, [Aib], Leu, Val or anacidic or neutral amino acid residue, or Arg or other basic amino acidresidue; X⁴⁰ can be absent, or can be Phe, [AMEF], Trp, [d-Phe] (alsodepicted as f), [hPhe], Tyr, [Aib], [pI-Phe] or a neutral or hydrophobicamino acid residue, or Glu or other acidic amino acid residue; X⁴¹ canbe absent, Lys, [NMeLys], [d-Lys] (also depicted as k), Gln, Glu, His,Ala, [Aib], Leu, Arg or a basic, or acidic, or neutral amino acidresidue; X⁴² can be absent, or can be Ala, Glu, [Aib], Pro, Cys or anacidic or neutral amino acid residue; X⁴³ can be absent, or can be Gly,Glu, Ala, [Aib], Asn, Gln, Trp, Cys or a neutral or acidic amino acidresidue, or His, Arg or other basic amino acid residue; X⁴⁴ can beabsent, Lys, His, [d-Lys] (also depicted as k), [Orn], Ala, Phe, Asn,Gln, Arg, Trp, Tyr or a basic or neutral amino acid residue, or-Gly-Ser, -Gly-Gly-Gly-Ser, -His-His-His-His-His, or-His-His-His-His-His-His, or Glu or other acidic amino acid; whereinonly 11 amino acid residues can be absent at the same time.

In one aspect of the invention, wherein X¹ and X⁵ may optionally formdisulfide bonds. In another aspect, X¹⁴ and X³² may optionally formdisulfide bonds. The invention further provides PAC1 antagonisticpeptides, wherein the amino-terminal residue may be optionallyacetylated; and the carboxy-terminal residue may be optionally amidated.

The invention provides peptides or a pharmaceutically acceptable saltthereof, wherein X⁰ is G, acetyl, A, M, S, G-S-, [Pra], G-M-, or absent;for example, G or absent. It also provides that X¹ can be C or S. In oneexample, X² can be D or E. In another aspect, X³ can be A, P or [Aib].In the next example, X⁴ can be T, Y, S, V or absent. In a furtherexample, X⁵ is C or S. In one aspect, X¹ and X⁵ can be both C andconnected by a disulfide bond. In one aspect, X⁶ is Q, H, F, K, [Cit],E, N or S. In another aspect, X⁷ can be F or Y. The invention providesthat X⁸ can be R or K, or that X⁹ is K or R. In one example, X¹⁰ can beA, G, V, C or L. In another example, X¹¹ can be I, [Aib] or L. In afurther example, X¹² can be D, V or E. Further, X¹³ can be D, E, A or[Aib]. The invention provides peptides, wherein X¹⁴ can be C or W. X¹⁵can be Q, D, K, A, L, Y, F, V, W, S, M, H or I. In one example, X¹⁵ canbe A, D, V, or L; and X¹⁶ can be K, R, Y, V, W, I, L, T, F, M, T, [Pra],W, Q, [Aib] or [hGlu], H, G, in particular, R, K, or L. In anotherexample, X¹⁷ can be Q, A, R, [Aib], [Cit], W, L, S, Y, F, W, V, D, I, Kor V; or, for example, Q or R. In a further aspect, X¹⁸ can be A, S,[Pra], K, E, L or [Aib]. In another example, X¹⁹ can be H, W, R, K, Y,M, A, I, L, S, Q, V, F or absent, in particular, H, W or Y. In a furtherexample, X²⁰ can be H, Y, [Aib], A, or absent.

The peptides of the invention provided herein include examples whereinX²¹ can be S, A, M, [Aib] or V. In one example, X²² can be N, Q, E, H orabsent, in particular N or absent. In another example, X²³ can be V, G,K, E, T or absent, for example, V or absent. In another example, X²⁴ canbe P, [DHP], R, [Sar], [Hyp], [Pip] or absent. For example, X²¹ can beG, [Pra], A, Y, or absent. In a further aspect of the invention, X²⁶ canbe N, -LQTSV-, Q or absent. The invention provides that X²⁷ can be S,[Aib], A, G, D or absent. The invention further provides that X²⁸ can beV, K, T, A or absent. In one example, X²⁹ is F, W, Y, [Aib] or absent.In another example, X³⁰ can be K, [Aib], A, [d-a] (also depicted as a)or E.

The invention provides PAC1 antagonist peptides, wherein X³¹ can be E,S, A, L, [Aib], T, V or K. in one example, X³² can be C or R. In afollowing example, X³³ can be M, [SeMet], L, F, I, S, [Nle] or V. In anext example, X³⁴ can be K, V, L, [Aib], A or E. in one aspect, X³⁵ canbe Q, E, R, F, [Aib], A, T, N or P. In one aspect, X³⁶ can be K, R, H,L, C, A, [d-k] (also depicted as k), [Orn], Q, [Aib], E, G or [NMeLys].In another aspect, X³⁷ can be K, R, F, A, [Aib] or Q. In a furtheraspect, X³⁸ can be K, R, L, F, T, N, [Aib], A, [Orn], E, Q, G, [d-k](also depicted as k) or [NMeLys]. In a still further example, X³⁹ can beE, R, L, V, A or [Aib]. In one aspect, X⁴⁰ can be F, W, Y, [d-f] (alsodepicted as f), [AMEF], [Aib], [hPh], E or [pI-Phe], in particular, W orF. In another aspect, X⁴¹ can be K, L, R, Q, [d-k] (also depicted as k),[Aib], [NMeLys], E, A, H or absent. In a further aspect, X⁴² can be A,[Aib], C, E, P or absent. In a further aspect, X⁴³ can be G, N, R, H, A,Q, W, [Aib], E or absent. In one aspect, X⁴⁴ can be K, H, Y, N, -His₆,-G₃S, R, W, A, Q, F, -His₅, E, -G-S, [d-k] (also depicted as k), [Orn]or absent, in particular, H, Y or absent. The invention providespeptides wherein X¹⁴ and X³² can be both C and connected by a disulfidebond.

The peptides contemplated by the invention include PAC1 antagonistcpeptides of Formula 1, wherein the N-terminus of the peptide isacetylated and the C-terminus of the peptide is amidated. In one aspect,a peptide or a pharmaceutically acceptable salt thereof is of SEQ IDNO:1-SEQ ID NO:451. The invention further provides compositionscomprising the peptide of the invention, or a pharmaceuticallyacceptable sale thereof and a pharmaceutically acceptable excipient.

The invention also contemplates the methods of treatment of migraineusing any of the peptides of the invention, or a pharmaceuticallyacceptable salt thereof. In one aspect, the invention includes themethod of any as above, wherein the method comprises acute treatment.

DETAILED DESCRIPTION

This invention disclosure generally relates to novel synthetic peptidesthat are PAC1 receptor antagonists and their use for treating migraine,especially acute migraine.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well-known and commonly used in the art. Themethods and techniques of the present application are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001), Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates (1992), and Harlow and LaneAntibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990), which are incorporated herein byreference. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The terminology used in connection with,and the laboratory procedures and techniques of, analytical chemistry,synthetic organic chemistry, and medicinal and pharmaceutical chemistrydescribed herein are those well-known and commonly used in the art.Standard techniques can be used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentagesmeans±10%.

Definitions

The terms “polypeptide” or “protein” are used interchangeably herein torefer to a polymer of amino acid residues. The terms also apply to aminoacid polymers in which one or more amino acid residues is an analog ormimetic of a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers. The terms can also encompassamino acid polymers that have been modified, e.g., by the addition ofcarbohydrate residues to form glycoproteins, or phosphorylated.Polypeptides and proteins can be produced by a naturally-occurring andnon-recombinant cell; or by a genetically-engineered or recombinantcell, and comprise molecules having the amino acid sequence of thenative protein, or molecules having deletions from, additions to, and/orsubstitutions of one or more amino acids of the native sequence. Theterms “polypeptide” and “protein” specifically encompass antibodies,e.g., anti-PAC1 antibodies (aka PAC1 antibodies), PAC1 binding proteins,antibodies, or sequences that have deletions from, additions to, and/orsubstitutions of one or more amino acids of an antigen-binding protein.The term “polypeptide fragment” refers to a polypeptide that has anamino-terminal deletion, a carboxyl-terminal deletion, and/or aninternal deletion as compared with the full-length protein. Suchfragments may also contain modified amino acids as compared with thefull-length protein.

The term “isolated protein” or “isolated polypeptide” means that asubject protein or polypeptide is free of most other proteins with whichit would normally be found and has been separated from at least about 50percent of polynucleotides, lipids, carbohydrates, or other materialswith which it is associated in nature. Typically, an “isolated protein”or “isolated polypeptide” constitutes at least about 5%, at least about10%, at least about 25%, or at least about 50% of a given sample.Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or anycombination thereof may encode such an isolated protein.

Preferably, the isolated protein polypeptide or antibody issubstantially free from other proteins or other polypeptides or othercontaminants that are found in its natural environment that wouldinterfere with its therapeutic, diagnostic, prophylactic, research orother use.

The terms “human PAC1”, “human PAC₁”, “hPAC1” and “hPAC₁”, “human PAC1receptor”, “human PAC₁ receptor”, “hPAC1 receptor” and “hPAC₁ receptor”are used interchangeably and refer to the human pituitary adenylatecyclase-activating polypeptide type I receptor. hPAC1 is a 468 aminoacid protein designated as P41586 (PACR_HUMAN) in theUniProtKB/Swiss-Prot database and is encoded by the ADCYAP1R1 gene.PACAP-27 and PACAP-38 are the principal endogenous agonists of PAC1.Unless otherwise specified or clear from the context in which the termis used, “PAC1” refers to human PAC1.

The terms “fragment,” “derivative,” and “variant,” when referring to thepolypeptides of the invention, means fragments, derivatives, andvariants of the polypeptides which retain substantially the samebiological function or activity as such polypeptides, as describedfurther below. The fragment, derivative, or variant of the polypeptidesof the present invention may be (i) one in which one or more of theamino acid residues are substituted with a conserved or non-conservedamino acid residue (e.g., a conserved amino acid residue) and suchsubstituted amino acid residue may or may not be one encoded by thegenetic code, or (ii) one in which one or more of the amino acidresidues includes a substituent group, or (iii) one in which the maturepolypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (e.g., polyethyleneglycol orPEG), or (iv) one in which the additional amino acids are fused to themature polypeptide, such as a leader or secretory sequence or a sequencewhich is employed for purification of the mature polypeptide or apropolypeptide sequence, or (v) one in which the polypeptide sequence isfused with a larger polypeptide (e.g., human albumin, an antibody or Fc,for increased duration of effect). Such fragments, derivatives, andvariants and analogs are deemed to be within the scope of those skilledin the art from the teachings herein.

“Functional equivalent” and “substantially the same biological functionor activity” each means that degree of biological activity that iswithin about 30% to about 100% or more of that biological activitydemonstrated by the polypeptide to which it is being compared when thebiological activity of each polypeptide is determined by the sameprocedure.

A “variant” of a polypeptide comprises an amino acid sequence whereinone or more amino acid residues are inserted into, deleted from and/orsubstituted into the amino acid sequence relative to another polypeptidesequence. Variants include polypeptides that differ in amino acidsequence due to mutagenesis. Variants that function as PAC1 antagonistscan be identified by screening combinatorial libraries of mutants, forexample truncation mutants, of the polypeptides of this invention forPAC1 antagonist activity.

A “derivative” of a polypeptide is a polypeptide that has beenchemically modified in some manner distinct from insertion, deletion, orsubstitution variants, e.g., via conjugation to another chemical moiety.A derivative includes all modifications to the polypeptide whichsubstantially preserve the functions disclosed herein and includeadditional structure and attendant function (e.g., PEGylatedpolypeptides which have greater half-life), fusion polypeptides whichconfer targeting specificity, or an additional activity such as toxicityto an intended target. The derivatives of the present invention maycontain conservative amino acid substitutions (defined further below)made at one or more predicted, e.g., nonessential amino acid residues. A“nonessential” amino acid residue is a residue that can be altered fromthe wild-type sequence of a protein without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

A “fragment” is a portion of the polypeptide which retains substantiallysimilar functional activity, as described in the models disclosedherein. Fragments, or biologically active portions include polypeptidefragments suitable for use as a medicament, to generate antibodies, as aresearch reagent, and the like. Fragments include peptides comprisingamino acid sequences sufficiently similar to or derived from the aminoacid sequences of a polypeptide of this invention and exhibiting atleast one activity of that polypeptide, but which include fewer aminoacids than the full-length polypeptides disclosed herein.

Typically, biologically active portions comprise a domain or motif withat least one activity of the polypeptide. A biologically active portionof a polypeptide can be a peptide which is, for example, five or moreamino acids in length. Such biologically active portions can be preparedsynthetically or by recombinant techniques and can be evaluated for oneor more of the functional activities of a polypeptide of this inventionby means disclosed herein and/or well known in the art.

An “analog” includes a pro-polypeptide which includes within it, theamino acid sequence of the polypeptide of this invention. The activepolypeptide of this invention can be cleaved from the additional aminoacids that complete the pro-polypeptide molecule by natural, in vivoprocesses or by procedures well known in the art such as by enzymatic orchemical cleavage.

The polypeptides of the present invention may be recombinantpolypeptides, natural purified polypeptides, or synthetic polypeptides.

The term “naturally occurring” as used throughout the specification inconnection with biological materials such as polypeptides, nucleicacids, host cells, and the like, refers to materials which are found innature.

The invention also provides chimeric or fusion polypeptides. Thetargeting sequence is designed to localize the delivery of thepolypeptide to minimize potential side effects. The polypeptides of thisinvention can be composed of amino acids joined to each other by peptidebonds or modified peptide bonds (i.e., peptide isosteres), and maycontain amino acids other than the 20 gene-encoded amino acids. Thepolypeptides may be modified by either natural processes, such asposttranslational processing, or by chemical modification techniqueswhich are well known in the art.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formulation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, PEGylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination (see, e.g.,Proteins, Structure and Molecular Properties, 2nd ed., T. E. Creighton,W. H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, ed., Academic Press, New York,pp. 1-12 (1983); Seifter, et al., Meth. Enzymol 182:626-646, 1990;Rattan, et al., Ann. N.Y. Acad. Sci. 663:48-62, 1992).

In the case of PEGylation, the fusion of the peptide of the invention toPEG may be accomplished by any means known to one skilled in the art.For example, PEGylation may be accomplished by first introducing acysteine mutation into the peptide to provide a linker upon which toattach the PEG, followed by site-specific derivatization withPEG-maleimide. Alternatively, the N-terminal modification mayincorporate a reactive moiety for coupling to PEG, as exemplified by theamine group, the mercapto group, or the carboxylate group of theN-terminal modifying compounds disclosed above. For example, PEGylationmay be accomplished by first introducing a mercapto moiety into thepolypeptide via the N-terminal modifying group to provide a linker uponwhich to attach the PEG, followed by site-specific derivatization withmethoxy-PEG-maleimide reagents supplied by, for example, either NektarTherapeutics (San Carlos, Calif., USA) and/or NOF (Tokyo, Japan). Inaddition to maleimide, numerous Cys reactive groups are known to thoseskilled in the art of protein cross-linking, such as the use of alkylhalides and vinyl sulfones (see, e.g., Proteins, Structure and MolecularProperties, 2nd ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993). In addition, the PEG could be introduced by directattachment to the C-terminal carboxylate group, or to an internal aminoacid such as Cys, Lys, Asp, or Glu or to unnatural amino acids thatcontain similar reactive sidechain moieties.

Various size PEG groups can be used, as exemplified but not limited to,PEG polymers of from about 5 kDa to about 43 kDa. The PEG modificationmay include a single, linear PEG. For example, linear 5, 20, or 30 kDaPEGs that are attached to maleidmide or other cross-linking groups areavailable from Nektar and/or NOF. Also, the modification may involvebranched PEGs that contain two or more PEG polymer chains that areattached to maleimide or other cross-linking groups are available fromNektar and NOF.

The linker between the PEG and the peptide cross-linking group can bevaried. For example, the commercially available thiol-reactive 40 kDaPEG (mPEG2-MAL) from Nektar (Huntsville, Ala.) employs a maleimide groupfor conjugation to Cys, and the maleimide group is attached to the PEGvia a linker that contains a Lys. As a second example, the commerciallyavailable thiol-reactive 43 kDa PEG (GL2-400MA) from NOF employs amaleimide group for conjugation to Cys, and the maleimide group isattached to the PEG via a bi-substituted alkane linker. In addition, thePEG polymer can be attached directly to the maleimide, as exemplified byPEG reagents of molecular-weight 5 and 20 kDa available from NektarTherapeutics (Huntsville, Ala.).

The polypeptides of the present invention include, for example, thepolypeptides of SEQ ID NOs: 1 through 451), as well as those sequenceshaving insubstantial variations in sequence from them. An “insubstantialvariation” would include any sequence addition, substitution, ordeletion variant that maintains substantially at least one biologicalfunction of the polypeptides of this invention, such as PAC1 antagonistactivity demonstrated herein. These functional equivalents may include,for example, polypeptides which have at least about 90% identity to thepolypeptides of the invention, or at least 95% identity to thepolypeptides of the invention, or at least 97% identity to thepolypeptides of the invention, and also include portions of suchpolypeptides having substantially the same biological activity. However,any polypeptide having insubstantial variation in amino acid sequencefrom the polypeptides of the invention that demonstrates functionalequivalency as described further herein is included in the descriptionof the present invention.

Certain terms used throughout this specification are defined below. Thesingle letter abbreviation for a particular amino acid, itscorresponding amino acid, and three letter abbreviation are as follows:A, alanine (ala); C, cysteine (cys); D, aspartic acid (asp); E, glutamicacid (glu); F, phenylalanine (phe); G, glycine (gly); H, histidine(his); I, isoleucine (ile); K, lycine (lys); L, leucine (leu); M,methionine (met); N, asparagine (asn); P, proline (pro); Q, glutamine(gin); R, arginine (arg); S, serine (ser); T, threonine (thr); V, valine(val); W, tryptophan (trp); Y, tyrosine (tyr). In addition, thefollowing abbreviations have been used: a or [d-a] or [d-Ala],D-alanine; [Aib], 2-aminoisobutyric acid; [AMEF],alpha-methyl-phenylalanine; [Cit], citrulline; [DHP],3,4-Dehydro-Proline; f, or [d-f] or [d-Phe], D-phenylalanine; k, or[d-k] or [d-Lys], D-lysine; G-M, glycine-methionine fragment; G-S,glycine-serine fragment;; G₃S, glycine-glycine-glycine-serine; [hGlu],homoglutamic acid; His5,histidine-histidine-histidine-histidine-histidine; His6,histidine-histidine-histidine-histidine-histidine-histidine; [hPhe],homophenylalanine; [Hyp], 4-hydroxyproline (or hydroxyproline); LQTSV,leucine-glutamine-threonine-serine-valine; [Nle], norleucine; [NMeLys],N^(□)-methyllysine [Orn], ornithin; [Pip], pipecolic acid; [pI-Phe],para-iodophenyalanine (or 4-iodophenylalanine); [Pra], propargylglycineor 2-propargylglycine; [Sar], sarcosine; [SeMet], selenomethionine.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.Such conservative substitutions include those described above and byDayhoff (The Atlas of Protein Sequence and Structure 5, 1978), and byArgos (EMBO J. 8:779-785, 1989). For example, amino acids belonging toone of the following groups represent conservative changes:

-   -   ala, pro, gly, gln, asn, ser, thr;    -   cys, ser, tyr, thr;    -   val, ile, leu, met, ala, phe;    -   lys, arg, his;    -   phe, tyr, trp, his; and    -   asp, glu.

Also provided are related compounds within the understanding of thosewith skill in the art, such as chemical mimetics, organomimetics, orpeptidomimetics. As used herein, the terms “mimetic,” “peptide mimetic,”“peptidomimetic,” “organomimetic,” and “chemical mimetic” are intendedto encompass peptide derivatives, peptide analogs, and chemicalcompounds having an arrangement of atoms in a three-dimensionalorientation that is equivalent to that of a peptide of the presentinvention. It is understood that the phrase “equivalent to” as usedherein is intended to encompass compounds having substitution(s) ofcertain atoms, or chemical moieties in said peptide, having bondlengths, bond angles, and arrangements in the mimetic compound thatproduce the same or sufficiently similar arrangement or orientation ofsaid atoms and moieties to have the biological function of the peptidesof the invention. In the peptide mimetics of the invention, thethree-dimensional arrangement of the chemical constituents isstructurally and/or functionally equivalent to the three-dimensionalarrangement of the peptide backbone and component amino acid sidechainsin the peptide, resulting in such peptido-, organo-, and chemicalmimetics of the peptides of the invention having substantial biologicalactivity. These terms are used according to the understanding in theart, as illustrated, for example, by Fauchere, (Adv. Drug Res. 15:29,1986); Veber & Freidinger, (TINS p. 392, 1985); and Evans, et al., (J.Med. Chem. 30:1229, 1987).

It is understood that a pharmacophore exists for the biological activityof each peptide of the invention. A pharmacophore is understood in theart as comprising an idealized, three-dimensional definition of thestructural requirements for biological activity. Peptido-, organo-, andchemical mimetics may be designed to fit each pharmacophore with currentcomputer modeling software (computer aided drug design). Said mimeticsmay be produced by structure-function analysis, based on the positionalinformation from the substituent atoms in the peptides of the invention.

Peptides as provided by the invention can be advantageously synthesizedby any of the chemical synthesis techniques known in the art,particularly solid-phase synthesis techniques, for example, usingcommercially-available automated peptide synthesizers. The mimetics ofthe present invention can be synthesized by solid phase or solutionphase methods conventionally used for the synthesis of peptides (see,e.g., Merrifield, J. Am. Chem. Soc. 85:2149-54, 1963; Carpino, Acc.Chem. Res. 6:191-98, 1973; Birr, Aspects of the Merrifield PeptideSynthesis, Springer-Verlag: Heidelberg, 1978; The Peptides: Analysis,Synthesis, Biology, Vols. 1, 2, 3, and 5, (Gross & Meinhofer, eds.),Academic Press: New York, 1979; Stewart, et al., Solid PhasePeptideSynthesis, 2nd. ed., Pierce Chem. Co.: Rockford, Ill., 1984;Kent, Ann. Rev. Biochem. 57:957-89, 1988; and Gregg, et al., Int. J.Peptide Protein Res. 55:161-214, 1990, which are incorporated herein byreference in their entirety.)

The solid phase methodology may also be utilized. Briefly, anN-protected C-terminal amino acid residue is linked to an insolublesupport such as divinylbenzene cross-linked polystyrene, polyacrylamideresin, Kieselguhr/polyamide (pepsyn K), controlled pore glass,cellulose, polypropylene membranes, acrylic acid-coated polyethylenerods, or the like. Cycles of deprotection, neutralization, and couplingof successive protected amino acid derivatives are used to link theamino acids from the C-terminus according to the amino acid sequence.For some synthetic peptides, an FMOC strategy using an acid-sensitiveresin may be used. Examples of solid supports in this regard aredivinylbenzene cross-linked polystyrene resins, which are commerciallyavailable in a variety of functionalized forms, including chloromethylresin, hydroxymethyl resin, paraacetamidomethyl resin, benzhydrylamine(BHA) resin, 4-methylbenzhydrylamine (MBHA) resin, oxime resins,4-alkoxybenzyl alcohol resin (Wang resin),4-(2′,4′-dimethoxyphenylaminomethyl)-phenoxymethyl resin,2,4-dimethoxybenzhydryl-amine resin, and4-(2′,4′-dimethoxyphenyl-FMOC-amino-methyl)-phenoxyacetamidonorleucyl-MBHAresin (Rink amide MBHA resin). In addition, acid-sensitive resins alsoprovide C-terminal acids, if desired. One protecting group for alphaamino acids could be base-labile 9-fluorenylmethoxy-carbonyl (FMOC).

Suitable protecting groups for the side chain functionalities of aminoacids chemically compatible with BOC (t-butyloxycarbonyl) and FMOCgroups are well known in the art. When using FMOC chemistry, thefollowing protected amino acid derivatives are preferred:FMOC-Cys(Trit), FMOC-Ser(But), FMOC-Asn(Trit), FMOC-Leu, FMOC-Thr(Trit),FMOC-Val, FMOC-Gly, FMOC-Lys(Boc), FMOC-Gln(Trit), FMOC-Glu(OBut),FMOC-His(Trit), FMOC-Tyr(But), FMOC-Arg(PMC(2,2,5,7,8-pentamethylchroman-6-sulfonyl)), FMOC-Arg(BOC)₂, FMOC-Pro,and FMOC-Trp(BOC). The amino acid residues may be coupled by using avariety of coupling agents and chemistries known in the art, such asdirect coupling with DIC (diisopropyl-carbodiimide), or Oxyma (ethylcyanohydroxyiminoacetate) with DIC, or DCC (dicyclohexylcarbodiimide),BOP (benzotriazolyl-N-oxytrisdimethylaminophosphoniumhexa-fluorophosphate), PyBOP(benzotriazole-1-yl-oxy-tris-pyrrolidinophosphoniumhexafluoro-phosphate), PyBrOP (bromo-tris-pyrrolidinophosphoniumhexafluorophosphate); via performed symmetrical anhydrides; via activeesters such as pentafluorophenyl esters; or via performed HOBt(1-hydroxybenzotriazole) active esters or by using FMOC-amino acidfluoride and chlorides or by using FMOC-amino acid-N-carboxy anhydrides.Activation with HBTU(2-(1H-benzotriazole-1-yl),1,1,3,3-tetramethyluroniumhexafluorophosphate) or HATU (2-(1H-7-aza-benzotriazole-1-yl),1,1,3,3-tetramethyluronium hexafluoro-phosphate) in the presence of HOBtor HOAt (7-azahydroxybenztriazole) or TATU(0-(7-Azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate) can be used.

The solid phase method may be carried out manually, or automatedsynthesis on a commercially available peptide synthesizer (e.g., AppliedBiosystems 431A or the like; Applied Biosystems, Foster City, Calif.)may be used. In a typical synthesis, the first (C-terminal) amino acidis loaded on the chlorotrityl resin. Successive deprotection (with 20%piperidine/NMP (N-methylpyrrolidone)) and coupling cycles according toABI FastMoc protocols (Applied Biosystems) may be used to generate thepeptide sequence. Double and triple coupling, with capping by aceticanhydride, may also be used.

The synthetic mimetic peptide may be cleaved from the resin anddeprotected by treatment with TFA (trifluoroacetic acid) containingappropriate scavengers. Many such cleavage reagents, such as Reagent K(0.75 g crystalline phenol, 0.25 mL ethanedithiol, 0.5 mL thioanisole,0.5 mL deionized water, 10 mL TFA) and others, may be used. The peptideis separated from the resin by filtration and isolated by etherprecipitation. Further purification may be achieved by conventionalmethods, such as gel filtration and reverse phase HPLC (high performanceliquid chromatography). Synthetic mimetics according to the presentinvention may be in the form of pharmaceutically acceptable salts,especially base-addition salts including salts of organic bases andinorganic bases. The base-addition salts of the acidic amino acidresidues are prepared by treatment of the peptide with the appropriatebase or inorganic base, according to procedures well known to thoseskilled in the art, or the desired salt may be obtained directly bylyophilization of the appropriate base.

Skilled in the art will recognize that peptides of the invention may bemodified by a variety of chemical techniques to produce peptides havingessentially the same activity as the unmodified peptides of theinvention, and optionally having other desirable properties. In oneaspect, carboxylic acid groups of the peptide may be provided in theform of a salt of a pharmaceutically-acceptable cation. Amino groupswithin the peptide may be in the form of a pharmaceutically-acceptableacid addition salt, such as the HCl, HBr, acetic, benzoic, toluenesulfonic, maleic, tartaric, and other organic salts, or may be convertedto an amide. Thiols may be protected with any one of a number ofwell-recognized protecting groups, such as acetamide groups. Thoseskilled in the art will also recognize methods for introducing cyclicstructures into the peptides of this invention so that the nativebinding configuration will be more nearly approximated. For example, acarboxyl terminal or amino terminal cysteine residue may be added to thepeptide, so that when oxidized the peptide will contain a disulfidebond, thereby generating a cyclic peptide. Other peptide cyclizingmethods include the formation of thioethers and carboxyl- andamino-terminal amides and esters.

Specifically, a variety of techniques are available for constructingpeptide derivatives and analogs with the same or similar desiredbiological activity as the corresponding peptide compound but with morefavorable activity than the peptide with respect to solubility,stability, and susceptibility to hydrolysis and proteolysis. Suchderivatives and analogs include peptides modified at the N-terminalamino group, the C-terminal carboxyl group, and/or changing one or moreof the amido linkages in the peptide to a non-amido linkage. It will beunderstood that two or more such modifications may be coupled in onepeptide mimetic structure (e.g., modification at the C-terminal carboxylgroup and inclusion of a —CH₂— carbamate linkage between two amino acidsin the peptide).

Amino terminus modifications include alkylating, acetylating, adding acarbobenzoyl group, forming a succinimide group, and attaching one ormore amino acids. Specifically, the N-terminal amino group may bereacted to form an amide group of the formula RC(O)NH— where R is alkyl,for example, lower alkyl, and is added by reaction with an acid halide,RC(O)Cl or acid anhydride. Typically, the reaction can be conducted byadding about equimolar or excess amounts (e.g., about 5 equivalents) ofan acid halide to the peptide in an inert diluent (e.g.,dichloromethane) preferably containing an excess (e.g., about 10equivalents) of a tertiary amine, such as diisopropylethylamine, toscavenge the acid generated during reaction. Reaction conditions areotherwise conventional (e.g., room temperature for 30 minutes).Alkylation of the terminal amino to provide for a lower alkylN-substitution followed by reaction with an acid halide as describedabove will provide an N-alkyl amide group of the formula RC(O)NR—.Alternatively, the amino terminus may be covalently linked tosuccinimide group by reaction with succinic anhydride. An approximatelyequimolar amount or an excess of succinic anhydride (e.g., about 5equivalents) is used and the terminal amino group is converted to thesuccinimide by methods well known in the art including the use of anexcess (e.g., 10 equivalents) of a tertiary amine such asdiisopropylethylamine in a suitable inert solvent (e.g.,dichloromethane), as described in Wollenberg, et al., (U.S. Pat. No.4,612,132), and is incorporated herein by reference in its entirety. Itwill also be understood that the succinic group may be substituted with,for example, a C₂- through C₆-alkyl or —SR substituents, which areprepared in a conventional manner to provide for substituted succinimideat the N-terminus of the peptide. Such alkyl substituents may beprepared by reaction of a lower olefin (C₂- through C₆-alkyl) withmaleic anhydride in the manner described by Wollenberg, et al., supra.,and —SR substituents may be prepared by reaction of RSH with maleicanhydride where R is as defined above. In another aspect, the aminoterminus may be derivatized to form a benzyloxycarbonyl-NH— or asubstituted benzyloxycarbonyl-NH— group. This derivative may be producedby reaction with approximately an equivalent amount or an excess ofbenzyloxycarbonyl chloride (CBZ-Cl), or a substituted CBZ-Cl in asuitable inert diluent (e.g., dichloromethane) preferably containing atertiary amine to scavenge the acid generated during the reaction. Inyet another derivative, the N-terminus comprises a sulfonamide group byreaction with an equivalent amount or an excess (e.g., 5 equivalents) ofR—S(O)₂C1 in a suitable inert diluent (dichloromethane) to convert theterminal amine into a sulfonamide, where R is alkyl and preferably loweralkyl. In one aspect, the inert diluent contains excess tertiary amine(e.g., 10 equivalents) such as diisopropylethylamine, to scavenge theacid generated during reaction. Reaction conditions are otherwiseconventional (e.g., room temperature for 30 minutes). Carbamate groupsmay be produced at the amino terminus by reaction with an equivalentamount or an excess (e.g., 5 equivalents) of R—OC(O)Cl orR—OC(O)OC₆H₄-p-NO₂ in a suitable inert diluent (e.g., dichloromethane)to convert the terminal amine into a carbamate, where R is alkyl,preferably lower alkyl. For example, the inert diluent may contain anexcess (e.g., about 10 equivalents) of a tertiary amine, such asdiisopropylethylamine, to scavenge any acid generated during reaction.Reaction conditions are otherwise conventional (e.g., room temperaturefor 30 minutes). Urea groups may be formed at the amino terminus byreaction with an equivalent amount or an excess (e.g., 5 equivalents) ofR—N═C═O in a suitable inert diluent (e.g., dichloromethane) to convertthe terminal amine into a urea (i.e., RNHC(O)NH—) group where R is asdefined above. In one aspect, the inert diluent contains an excess(e.g., about 10 equivalents) of a tertiary amine, such asdiisopropylethylamine. Reaction conditions can be otherwise conventional(e.g., room temperature for about 30 minutes). Attachment of one or moreresidues at the amino terminus could be accomplished by any of thechemical synthesis techniques known in the art. Using solid phasesynthesis techniques, for example, additional residues could be appendedby continuing the cycles of deprotection, neutralization, and couplingof protected amino acid derivatives after installation of the aminoterminus of any sequence.

When preparing peptide mimetics, the C-terminal carboxyl group may beextended with additional amino acids. The extension could beaccomplished by any of the chemical synthesis techniques known in theart. Using solid phase synthesis techniques, for example, additionalresidues to the C-terminus could synthesized by adding the residues tothe resin first via cycles of deprotection, neutralization, and couplingof protected amino acid derivatives followed by installation of theamino acids that comprise any sequence.

In preparing peptide mimetics wherein the C-terminal carboxyl group maybe replaced by an ester (e.g., —C(O)OR where R is alkyl and, in oneaspect, lower alkyl), resins used to prepare the peptide acids may beemployed, and the side chain protected peptide may be cleaved with abase and the appropriate alcohol (e.g., methanol). Side chain protectinggroups may be removed in the usual fashion by treatment with hydrogenfluoride to obtain the desired ester. In preparing peptide mimeticswherein the C-terminal carboxyl group can be replaced by the amide—C(O)NR₃R₄, a benzhydrylamine resin may be used as the solid support forpeptide synthesis. Upon completion of the synthesis, hydrogen fluoridetreatment can be used to release the peptide from the support resultsdirectly in the free peptide amide (i.e., the C-terminus is —C(O)NH₂).Alternatively, use of the chloromethylated resin during peptidesynthesis coupled with reaction with ammonia to cleave the side chainprotected peptide from the support yields the free peptide amide, andreaction with an alkylamine or a dialkylamine yields a side chainprotected alkylamide or dialkylamide (i.e., the C-terminus can be—C(O)NRR, where R and R₁ are alkyl such as lower alkyl). Side chainprotection can be then removed in the usual way by treatment withhydrogen fluoride to give the free amides, alkylamides, ordialkylamides.

In another aspect, the C-terminal carboxyl group or a C-terminal estermay be induced to cyclize by displacement of the —OH or the ester (—OR)of the carboxyl group or ester, respectively, with the N-terminal aminogroup to form a cyclic peptide. For example, after synthesis andcleavage to give the peptide acid, the free acid can be converted insolution to an activated ester by an appropriate carboxyl groupactivator such as dicyclohexylcarbodiimide (DCC), for example, inmethylene chloride (CH₂Cl₂), dimethyl formamide (DMF), or mixturesthereof. The cyclic peptide can then be formed by displacement of theactivated ester with the N-terminal amine. Cyclization, rather thanpolymerization, may be enhanced by use of very dilute solutionsaccording to methods well known in the art.

Peptide mimetics can be structurally similar to the peptide of theinvention, but have one or more peptide linkages optionally replaced bya linkage selected from the group consisting of: —CH₂NH—, —CH₂S—,—CH₂CH₂—, —CH═CH— (in both cis and trans conformers), —COCH₂—,—CH(OH)CH₂—, and —CH₂SO—, by methods known in the art and furtherdescribed in the following references: Spatola, Chemistry andBiochemistry of Amino Acids, Peptides, and Proteins, (Weinstein, ed.),Marcel Dekker: New York, p. 267, 1983; Spatola, Peptide BackboneModifications 1:3, 1983; Morley, Trends Pharm. Sci. pp. 463-468, 1980;Hudson, et al., Int. J. Pept. Prot. Res. 14:177-185, 1979; Spatola, etal., Life Sci. 38:1243-1249, 1986; Hann, J. Chem. Soc. Perkin Trans.1307-314, 1982; Almquist, et al., J. Med. Chem. 23:1392-1398, 1980;Jennings-White, et al., Tetrahedron Lett. 23:2533, 1982; Szelke, et al.,EP045665A; Holladay, et al., Tetrahedron Lett. 24:4401-4404, 1983; andHruby, Life Sci. 31:189-199, 1982. Such peptide mimetics may havesignificant advantages over polypeptide embodiments, including, forexample, more economical to produce, having greater chemical stabilityor enhanced pharmacological properties (such as half-life, absorption,potency, efficacy, etc.), reduced antigenicity, and other properties.

Mimetic analogs of the peptides of the invention may also be obtainedusing the principles of conventional or rational drug design (see, e.g.,Andrews, et al., Proc. Alfred Benzon Symp. 28:145-165, 1990; McPherson,Eur. J. Biochem. 189:1-24, 1990; Hol, et al., in Molecular Recognition:Chemical and Biochemical Problems, (Roberts, ed.); Royal Society ofChemistry; pp. 84-93, 1989a; Hol, Arzneim-Forsch. 39:1016-1018, 1989b;Hol, Agnew Chem. Int. Ed. Engl. 25:767-778, 1986).

In accordance with the methods of conventional drug design, the desiredmimetic molecules may be obtained by randomly testing molecules whosestructures have an attribute in common with the structure of a “native”peptide. The quantitative contribution that results from a change in aparticular group of a binding molecule may be determined by measuringthe biological activity of the putative mimetic in comparison with theactivity of the peptide. In one embodiment of rational drug design, themimetic is designed to share an attribute of the most stablethree-dimensional conformation of the peptide.

Thus, for example, the mimetic may be designed to possess chemicalgroups that are oriented in a way sufficient to cause ionic,hydrophobic, or van der Waals interactions that are similar to thoseexhibited by the peptides of the invention, as disclosed herein.

One method for performing rational mimetic design employs a computersystem capable of forming a representation of the three-dimensionalstructure of the peptide. Molecular structures of the peptido-, organo-,and chemical mimetics of the peptides of the invention may be producedusing computer-assisted design programs commercially available in theart. Examples of such programs include SYBYL 6.5®, HQSAR™, and ALCHEMY2000™ (Tripos); GALAXY™ and AM2000™ (AM Technologies, Inc., San Antonio,Tex.); CATALYST™ and CERIUS™ (Molecular Simulations, Inc., San Diego,Calif.); CACHE PRODUCTS™, TSAR™, AMBER™, and CHEM-X™ (Oxford MolecularProducts, Oxford, Calif.) and CHEMBUILDER3D™ (Interactive Simulations,Inc., San Diego, Calif.), and Molecular Operating Environment (ChemicalComputing Group, Quebec, Canada), and Pymol, Maestro, Desmond &BioLuminate (Schrodinger, New York, N.Y.), and Discovery Studio (BIOVIA,San Diego, Calif.).

The peptido-, organo-, and chemical mimetics produced using the peptidesdisclosed herein using, for example, art-recognized molecular modelingprograms may be produced using conventional chemical synthetictechniques, methods designed to accommodate high throughput screening,including combinatorial chemistry methods. Combinatorial methods usefulin the production of the peptido-, organo-, and chemical mimetics of theinvention include phage display arrays, solid-phase synthesis, andcombinatorial chemistry arrays. Combinatorial chemistry production ofthe peptido-, organo-, and chemical mimetics of the invention may beproduced according to methods known in the art, including, but notlimited to, techniques disclosed in Terreft, (Combinatorial Chemistry,Oxford University Press, London, 1998); Look, et al., Bioorg. Med. Chem.Lett. 6:707-12, 1996; Ruhland, et al., J. Am. Chem. Soc. 118: 253-4,1996; Gordon, et al., Acc. Chem. Res. 29:144-54, 1996; Pavia, “TheChemical Generation of Molecular Diversity”, Network Science Center,www.netsci.org, 1995; Adnan, et al., “Solid Support CombinatorialChemistry in Lead Discovery and SAR Optimization,” Id., 1995; Davies andBriant, “Combinatorial Chemistry Library Design using PharmacophoreDiversity,” Id., 1995; Pavia, “Chemically Generated Screening Libraries:Present and Future,” Id., 1996; and U.S. Pat. Nos. 5,880,972; 5,463,564;5,331,573; and 5,573,905.

The newly synthesized polypeptides may be substantially purified bypreparative high performance liquid chromatography (see, e.g.,Creighton, Proteins: Structures And Molecular Principles, W H Freemanand Co., New York, N.Y., 1983). The composition of a syntheticpolypeptide of the present invention may be confirmed by amino acidanalysis or sequencing by, for example, the Edman degradation procedure(Creighton, supra). Additionally, any portion of the amino acid sequenceof the polypeptide may be altered during direct synthesis and/orcombined using chemical methods with sequences from other proteins toproduce a variant polypeptide or a fusion polypeptide.

The term “treating” refers to any indicia of success in the treatment oramelioration of an injury, pathology or condition, including anyobjective or subjective parameter such as abatement; remission;diminishing of symptoms or making the injury, pathology or conditionmore tolerable to the patient; slowing in the rate of degeneration ordecline; making the final point of degeneration less debilitating;improving a patient's physical or mental well-being. The treatment oramelioration of symptoms can be based on objective or subjectiveparameters; including the results of a physical examination,neuropsychiatric exams, and/or a psychiatric evaluation. For example,certain methods presented herein successfully treat migraine headacheseither prophylactically or as an acute treatment, decreasing thefrequency of migraine headaches, decreasing the severity of migraineheadaches, and/or ameliorating a symptom associated with migraineheadaches.

An “effective amount” is generally an amount sufficient to reduce theseverity and/or frequency of symptoms, eliminate the symptoms and/orunderlying cause, prevent the occurrence of symptoms and/or theirunderlying cause, and/or improve or remediate the damage that resultsfrom or is associated with migraine headache. In some embodiments, theeffective amount is a therapeutically effective amount or aprophylactically effective amount. A “therapeutically effective amount”is an amount sufficient to remedy a disease state (e.g. migraineheadache) or symptoms, particularly a state or symptoms associated withthe disease state, or otherwise prevent, hinder, retard or reverse theprogression of the disease state or any other undesirable symptomassociated with the disease in any way whatsoever. A “prophylacticallyeffective amount” is an amount of a pharmaceutical composition that,when administered to a subject, will have the intended prophylacticeffect, e.g., preventing or delaying the onset (or reoccurrence) ofmigraine headache, or reducing the likelihood of the onset (orreoccurrence) of migraine headache or migraine headache symptoms. Thefull therapeutic or prophylactic effect does not necessarily occur byadministration of one dose, and may occur only after administration of aseries of doses. Thus, a therapeutically or prophylactically effectiveamount may be administered in one or more administrations.

“Amino acid” includes its normal meaning in the art. The twentynaturally-occurring amino acids and their abbreviations followconventional usage. See, Immunology-A Synthesis, 2nd Edition, (E. S.Golub and D. R. Green, eds.), Sinauer Associates: Sunderland, Mass.(1991), incorporated herein by reference for any purpose. Stereoisomers(e.g., D-amino acids) of the twenty conventional amino acids, unnaturalamino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids,and other unconventional amino acids may also be suitable components forpolypeptides and are included in the phrase “amino acid.” Examples ofunconventional amino acids include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, σ-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright-hand direction is the carboxyl-terminal direction, in accordancewith standard usage and convention.

Naturally-occurring amino acids may be divided into classes based oncommon side chain properties:

1) hydrophobic: Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

3) acidic: Asp, Glu;

4) basic: His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro, Ala

6) aromatic: Trp, Tyr, Phe.

Conservative amino acid substitutions may involve exchange of a memberof one of these classes with another member of the same class.Conservative amino acid substitutions may encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

Non-conservative substitutions may involve the exchange of a member ofone of the above classes for a member from another class. Suchsubstituted residues may be introduced into regions of the antibody thatare homologous with human antibodies, or into the non-homologous regionsof the molecule.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. The hydropathicprofile of a protein is calculated by assigning each amino acid anumerical value (“hydropathy index”) and then repetitively averagingthese values along the peptide chain. Each amino acid has been assigneda hydropathic index on the basis of its hydrophobicity and chargecharacteristics. They are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic profile in conferring interactivebiological function on a protein is understood in the art (see, e.g.,Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certainamino acids may be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, in certainembodiments, the substitution of amino acids whose hydropathic indicesare within +2 is included. In some aspects, those which are within +1are included, and in other aspects, those within +0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, as inthe present case. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigen-binding or immunogenicity, that is, with a biological propertyof the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within +2 is included, in otherembodiments, those which are within +1 are included, and in still otherembodiments, those within +0.5 are included. In some instances, one mayalso identify epitopes from primary amino acid sequences on the basis ofhydrophilicity.

Exemplary conservative amino acid substitutions are set forth in Table1.

TABLE 1 Conservative Amino Acid Substitutions Original Residue ExemplarySubstitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn GluAsp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu MetLeu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile,Leu

A skilled artisan will be able to determine suitable variants ofpolypeptides as set forth herein using well-known techniques. Oneskilled in the art may identify suitable areas of the molecule that maybe changed without destroying activity by targeting regions not believedto be important for activity. The skilled artisan also will be able toidentify residues and portions of the molecules that are conserved amongsimilar polypeptides. In further embodiments, even areas that may beimportant for biological activity or for structure may be subject toconservative amino acid substitutions without destroying the biologicalactivity or without adversely affecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a protein that correspond toamino acid residues important for activity or structure in similarproteins. One skilled in the art may opt for chemically similar aminoacid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the 3-dimensional structure andamino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of an antibody with respectto its three dimensional structure. A skilled artisan may choose not tomake radical changes to amino acid residues predicted to be on thesurface of the protein, since such residues may be involved in importantinteractions with other molecules. Moreover, one skilled in the art maygenerate test variants containing a single amino acid substitution ateach desired amino acid residue. These variants can then be screenedusing assays for PAC1 antagonizing activity, (see examples below) thusyielding information regarding which amino acids can be changed andwhich must not be changed. In other words, based on information gatheredfrom such routine experiments, skilled artisan can readily determine theamino acid positions where further substitutions should be avoidedeither alone or in combination with other mutations.

Labels and Effector Groups

In some embodiments, the polypeptide comprises one or more labels. Theterm “labeling group” or “label” means any detectable label. Examples ofsuitable labeling groups include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent groups (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentgroups, biotinyl groups, or predetermined polypeptide epitopesrecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, the labeling group is coupled to the peptidevia spacer arms of various lengths to reduce potential steric hindrance.Various methods for labeling proteins are known in the art and may beused as is seen fit.

The term “effector group” means any group coupled to a peptide that actsas a cytotoxic agent. Examples for suitable effector groups areradioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I) Other suitable groups include toxins, therapeuticgroups, or chemotherapeutic groups.

Examples of suitable groups include calicheamicin, auristatins,geldanamycin and maytansine. In some embodiments, the effector group iscoupled to the antibody via spacer arms of various lengths to reducepotential steric hindrance.

In general, labels fall into a variety of classes, depending on theassay in which they are to be detected: a) isotopic labels, which may beradioactive or heavy isotopes; b) magnetic labels (e.g., magneticparticles); c) redox active moieties; d) optical dyes; enzymatic groups(e.g. horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase); e) biotinylated groups; and f) predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags, etc.). In some aspects, the labeling group iscoupled to the peptide via spacer arms of various lengths to reducepotential steric hindrance. Various methods for labeling proteins areknown in the art.

Specific labels include optical dyes, including, but not limited to,chromophores, phosphors and fluorophores, with the latter being specificin many instances. Fluorophores can be either “small molecule” fluores,or proteinaceous fluores.

By “fluorescent label” is meant any molecule that may be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueJ, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705,Oregon green, the Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430,Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680),Cascade Blue, Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes,Eugene, Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.),Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). Suitableoptical dyes, including fluorophores, are described in MOLECULAR PROBESHANDBOOK by Richard P. Haugland.

Suitable proteinaceous fluorescent labels also include, but are notlimited to, green fluorescent protein, including a Renilla, Ptilosarcus,or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805),EGFP (Clontech Labs., Inc., Genbank Accession Number U55762), bluefluorescent protein (BFP, Quantum Biotechnologies, Inc., Quebec, Canada;Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol.6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Labs.,Inc.), luciferase (Ichiki et al., 1993, J. Immunol. 150:5408-5417), Pgalactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. U.S.A.85:2603-2607) and Renilla (WO92/15673, WO95/07463, WO98/14605,WO98/26277, WO99/49019, U.S. Pat. No. 5,292,658, No. 5418155, No.5683888, No. 5741668, No. 5777079, No. 5804387, No. 5874304, No.5876995, No. 5925558).

In certain embodiments, the invention provides a composition (e.g. apharmaceutical composition) comprising one or a plurality of the PAC1antagonists of the invention together with pharmaceutically acceptablediluents, carriers, excipients, solubilizers, emulsifiers,preservatives, and/or adjuvants.

Pharmaceutical compositions of the invention include, but are notlimited to, liquid, frozen, and lyophilized compositions.“Pharmaceutically-acceptable” refers to molecules, compounds, andcompositions that are non-toxic to human recipients at the dosages andconcentrations employed and/or do not produce allergic or adversereactions when administered to humans. In some embodiments, thepharmaceutical composition may contain formulation materials formodifying, maintaining or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption or penetration of the composition.In such embodiments, suitable formulation materials include, but are notlimited to, amino acids (such as glycine, glutamine, asparagine,arginine or lysine); antimicrobials; antioxidants (such as ascorbicacid, sodium sulfite or sodium hydrogen-sulfite); buffers (such asborate, bicarbonate, Tris-HCl, citrates, phosphates or other organicacids); bulking agents (such as mannitol or glycine); chelating agents(such as ethylenediamine tetraacetic acid (EDTA)); complexing agents(such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. Methodsand suitable materials for formulating molecules for therapeutic use areknown in the pharmaceutical arts, and are described, for example, inREMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (A. R. Genrmo, ed.),1990, Mack Publishing Company.

Compositions, Methods of Delivery. Methods of Treatment

In some aspects, the pharmaceutical composition of the inventioncomprises a standard pharmaceutical carrier, such as a sterile phosphatebuffered saline solution, bacteriostatic water, and the like. A varietyof aqueous carriers may be used, e.g., water, buffered water, 0.4%saline, 0.3% glycine and the like, and may include other proteins forenhanced stability, such as albumin, lipoprotein, globulin, etc.,subjected to mild chemical modifications or the like.

Exemplary concentrations of the peptides of the invention in theformulation may range from about 0.1 mg/ml to about 200 mg/ml or fromabout 0.1 mg/mL to about 50 mg/mL, or from about 0.5 mg/mL to about 25mg/mL, or alternatively from about 2 mg/mL to about 10 mg/mL. An aqueousformulation of the antigen binding protein may be prepared in apH-buffered solution, for example, at pH ranging from about 4.0 to about7.5, or from about 4.8 to about 5.5, or alternatively about 5.0.Examples of buffers that are suitable for a pH within this range includeacetate (e.g. sodium acetate), succinate (such as sodium succinate),gluconate, histidine, citrate, hydrogen chloride, hydrogenchloride/sodium hydroxide, PBS and other organic acid buffers. Thebuffer concentration can be from about 1 mM to about 200 mM, or fromabout 10 mM to about 60 mM, depending, for example, on the buffer andthe desired isotonicity of the formulation.

A tonicity agent, which may also stabilize the antigen binding protein,may be included in the formulation. Exemplary tonicity agents includepolyols, such as mannitol, sucrose or trehalose. Preferably the aqueousformulation is isotonic, although hypertonic or hypotonic solutions maybe suitable. Exemplary concentrations of the polyol in the formulationmay range from about 1% to about 15% w/v.

A surfactant may also be added to the formulations of the invention toreduce aggregation of the formulated peptide and/or minimize theformation of particulates in the formulation and/or reduce adsorption.Exemplary surfactants include nonionic surfactants such as polysorbates(e.g. polysorbate 20 or polysorbate 80) or poloxamers (e.g. poloxamer188). Exemplary concentrations of surfactant may range from about 0.001%to about 0.5%, or from about 0.005% to about 0.2%, or alternatively fromabout 0.004% to about 0.01% w/v.

In one example, the formulation contains the above-identified agents(i.e. antigen binding protein, buffer, polyol and surfactant) and isessentially free of one or more preservatives, such as benzyl alcohol,phenol, m-cresol, chlorobutanol and benzethonium chloride. In anotherembodiment, a preservative may be included in the formulation, e.g., atconcentrations ranging from about 0.1% to about 2%, or alternativelyfrom about 0.5% to about 1%. One or more other pharmaceuticallyacceptable carriers, excipients or stabilizers such as those describedin REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (A. R. Genrmo,ed.), 1990, Mack Publishing Company, may be included in the formulationprovided that they do not adversely affect the desired characteristicsof the formulation.

Therapeutic formulations of the peptides of the invention can beprepared for storage by mixing the peptides having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (A. R.Genrmo, ed.), 1990, Mack Publishing Company), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, maltose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

For example, a suitable formulation of the claimed invention may containan isotonic buffer such as a phosphate, acetate, or TRIS buffer incombination with a tonicity agent, such as a polyol, sorbitol, sucroseor sodium chloride, which tonicifies and stabilizes. One example of sucha tonicity agent is 5% sorbitol or sucrose. In addition, the formulationcould optionally include a surfactant at 0.01% to 0.02% wt/vol, forexample, to prevent aggregation or improve stability. The pH of theformulation may range from 4.5-6.5 or 4.5 to 5.5. Other exemplarydescriptions of pharmaceutical formulations for PAC1 antagonist peptidesmay be found in US Patent Publication No. 2003/0113316 and U.S. Pat. No.6,171,586. Suspensions and crystal forms of the peptides are alsocontemplated. Methods to make suspensions and crystal forms are known toone of skill in the art.

The formulations to be used for in vivo administration must be sterile.The compositions of the invention may be sterilized by conventional,well known sterilization techniques. For example, sterilization isreadily accomplished by filtration through sterile filtration membranes.The resulting solutions may be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile solution prior to administration.

The process of freeze-drying is often employed to stabilize polypeptidesfor long-term storage, particularly when the polypeptide is relativelyunstable in liquid compositions. A lyophilization cycle is usuallycomposed of three steps: freezing, primary drying, and secondary drying(see Williams and Polli, Journal of Parenteral Science and Technology,Volume 38, Number 2, pages 48-59, 1984). In the freezing step, thesolution is cooled until it is adequately frozen. Bulk water in thesolution forms ice at this stage. The ice sublimes in the primary dryingstage, which is conducted by reducing chamber pressure below the vaporpressure of the ice, using a vacuum. Finally, sorbed or bound water isremoved at the secondary drying stage under reduced chamber pressure andan elevated shelf temperature. The process produces a material known asa lyophilized cake. Thereafter the cake can be reconstituted prior touse.

The standard reconstitution practice for lyophilized material is to addback a volume of pure water (typically equivalent to the volume removedduring lyophilization), although dilute solutions of antibacterialagents are sometimes used in the production of pharmaceuticals forparenteral administration (see Chen, Drug Development and IndustrialPharmacy, Volume 18: 1311-1354, 1992).

Excipients have been noted in some cases to act as stabilizers forfreeze-dried products (see Carpenter et al., Volume 74: 225-239, 1991).For example, known excipients include polyols (including mannitol,sorbitol and glycerol); sugars (including glucose and sucrose); andamino acids (including alanine, glycine and glutamic acid). In addition,polyols and sugars are also often used to protect polypeptides fromfreezing and drying-induced damage and to enhance the stability duringstorage in the dried state. In general, sugars, in particulardisaccharides, are effective in both the freeze-drying process andduring storage. Other classes of molecules, including mono- anddi-saccharides and polymers such as PVP, have also been reported asstabilizers of lyophilized products.

For injection, the pharmaceutical formulation and/or medicament of theinvention may be a powder suitable for reconstitution with anappropriate solution as described above. Examples of these include, butare not limited to, freeze dried, rotary dried or spray dried powders,amorphous powders, granules, precipitates, or particulates. Forinjection, the formulations may optionally contain stabilizers, pHmodifiers, surfactants, bioavailability modifiers and combinations ofthese.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antigen binding protein, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and y ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the Lupron Depot™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated polypeptides remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The formulations of the invention may be designed to be short-acting,fast-releasing, long-acting, or sustained-releasing as described herein.Thus, the pharmaceutical formulations may also be formulated forcontrolled release or for slow release.

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, general health conditions, sex, and diet of thesubject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant invention.

The peptides of the invention can be administered by any suitable means,including parenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local treatment, intralesionaladministration. Parenteral administration includes intravenous,intraarterial, intraperitoneal, intramuscular, intradermal orsubcutaneous administration. In addition, the peptides of the inventioncan be suitably administered by pulse infusion, particularly withdeclining doses of the peptides. In one aspect, the dosing can be givenby injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Otheradministration methods are contemplated, including topical, particularlytransdermal, transmucosal, rectal, oral or local administration e.g.through a catheter placed close to the desired site. The peptides of theinvention can be administered intravenously or subcutaneously in aphysiological solution at a dose ranging between 0.01 mg/kg to 100 mg/kgat a frequency ranging from daily to weekly to monthly (e.g. every day,every other day, every third day, or 2, 3, 4, 5, or 6 times per week),preferably a dose ranging from 0.1 to 45 mg/kg, 0.1 to 15 mg/kg or 0.1to 10 mg/kg at a frequency of once per week, once every two weeks, oronce a month.

The novel peptide PAC1 antagonists of the invention are useful fortreating or ameliorating a condition associated with the biologicalactivity of PACAP in a patient in need thereof. As used herein, the term“treating” or “treatment” is an intervention performed with theintention of preventing the development or altering the pathology of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already diagnosed with or suffering from the disorder orcondition as well as those in which the disorder or condition is to beprevented. “Treatment” includes any indicia of success in theamelioration of an injury, pathology or condition, including anyobjective or subjective parameter such as abatement, remission,diminishing of symptoms, or making the injury, pathology or conditionmore tolerable to the patient, slowing in the rate of degeneration ordecline, making the final point of degeneration less debilitating, orimproving a patient's physical or mental well-being. The treatment oramelioration of symptoms can be based on objective or subjectiveparameters, including the results of a physical examination,self-reporting by a patient, neuropsychiatric exams, and/or apsychiatric evaluation.

Accordingly, in some aspects, the present invention provides a methodfor treating or preventing a condition associated with the biologicalactivity of PACAP, such as deactivation of the PAC1 receptors, in apatient in need thereof, comprising administering to the patient aneffective amount of a novel peptide described herein. The term “patient”includes human patients. PACAP biological activity has been implicatedin various physiological processes, including cardiovascular function,metabolic and endocrine function, inflammation, stress response, andregulation of the autonomic nervous system, particularly the balancebetween the sympathetic and parasympathetic systems. See, e.g., Tanidaet al., Regulatory Peptides, Vol. 161: 73-80, 2010; Moody et al., Curr.Opin. Endocrinol. Diabetes Obes., Vol. 18: 61-67, 2011; and Hashimoto etal., Current Pharmaceutical Design, Vol. 17: 985-989, 2011.

An “effective amount” is generally an amount sufficient to reduce theseverity and/or frequency of symptoms, eliminate the symptoms and/orunderlying cause, prevent the occurrence of symptoms and/or theirunderlying cause, and/or improve or remediate the damage that resultsfrom or is associated with a particular condition (e.g. chronic pain,headache or migraine). In some embodiments, the effective amount is atherapeutically effective amount or a prophylactically effective amount.A “therapeutically effective amount” is an amount sufficient to remedy adisease state (e.g., a headache, migraine, or chronic pain) orsymptom(s), particularly a state or symptom(s) associated with thedisease state, or otherwise prevent, hinder, retard or reverse theprogression of the disease state or any other undesirable symptomassociated with the disease in any way whatsoever (i.e., that provides“therapeutic efficacy”). A “prophylactically effective amount” is anamount of a pharmaceutical composition that, when administered to asubject, will have the intended prophylactic effect, e.g., preventing ordelaying the onset (or reoccurrence) of the condition (e.g., headache ormigraine), or reducing the likelihood of the onset (or reoccurrence) ofthe condition (e.g., headache, migraine, or headache symptoms). The fulltherapeutic or prophylactic effect does not necessarily occur byadministration of one dose, and may occur only after administration of aseries of doses. Thus, a therapeutically or prophylactically effectiveamount may be administered in one or more administrations.

In certain aspects, the present invention provides a method forinhibiting activation of the PAC1 receptor in a patient having aheadache condition comprising administering to the patient an effectiveamount of a peptide of the invention. For example, the method may treator prevent symptoms of the headache condition in the patient.Accordingly, the present invention also includes a method for treatingor preventing a headache condition, particularly migraine headache, in apatient in need thereof comprising administering to the patient aneffective amount of a PAC1 inhibiting peptide described herein.

In some methods of the invention, the headache condition to be treated,prevented or ameliorated is migraine. Migraine headaches are recurrentheadaches lasting about 4 to about 72 hours that are characterized byunilateral, pulsating, and/or moderate to severe pain and/or pain thatis exacerbated by physical activity. Migraine headaches are oftenaccompanied by nausea, vomiting, and/or sensitivity to light(photophobia), sound (phonophobia), or smell. In some patients, an auraprecedes the onset of the migraine headache. The aura is typically avisual, sensory, language, or motor disturbance that signals theheadache will soon occur. The methods described herein prevent, treat,or ameliorate one or more symptoms of migraine headaches with andwithout aura in human patients.

PACAP38, through activation of its receptors, induces vasodilation,particularly vasodilation of the dura vasculature (Schytz et al.,Neurotherapeutics, Vol. 7(2):191-196, 2010). The PACAP38/PAC1 receptorsignaling cascade, in particular, has been implicated in migrainepathophysiology (Amin et al., Brain, Vol. 137: 779-794, 2014). Infusionof PACAP38, which has a higher affinity for the PAC1 receptor than theVPAC1 and VPAC2 receptors, causes migraine-like headache in migrainepatients (Schytz et al., Brain 132:16-25, 2009; Amin et al., Brain, Vol.137: 779-794, 2014). In addition, PACAP38 levels are elevated in cranialcirculation in patients experiencing a migraine attack, and the PACAP38levels are reduced following treatment of the migraine symptoms withtriptans (Tuka et al., Cephalalgia, Vol. 33, 1085-1095, 2013; Zagami etal., Ann. Clin. Transl. Neurol., Vol. 1: 1036-1040, 2014). These reportssuggest that endogenous release of PACAP38 is an important trigger ofmigraine headache and its effects are primarily mediated throughactivation of the PAC1 receptor.

In some aspects of the invention, the patients to be treated accordingto the methods of the invention have, suffer from, or are diagnosed withepisodic migraine. Episodic or acute migraine is diagnosed when patientswith a history of migraine (e.g. at least five lifetime attacks ofmigraine headache) have 14 or fewer migraine headache days per month. A“migraine headache day” includes any calendar day during which a patientexperiences the onset, continuation, or recurrence of a “migraineheadache” with or without aura lasting greater than 30 minutes. A“migraine headache” is a headache associated with nausea or vomiting orsensitivity to light or sound and/or a headache characterized by atleast two of the following pain features: unilateral pain, throbbingpain, moderate to severe pain intensity, or pain exacerbated by physicalactivity. In certain embodiments, patients having, suffering from, ordiagnosed with episodic migraine have at least four, but less than 15migraine headache days per month on average. In related embodiments,patients having, suffering from, or diagnosed with episodic migrainehave fewer than 15 headache days per month on average. As used herein, a“headache day” is any calendar day in which the patient experiences amigraine headache as defined herein or any headache that lasts greaterthan 30 minutes or requires acute headache treatment.

In other aspects, the present invention provides a method for treatingor ameliorating cluster headache in a patient in need thereof comprisingadministering to the patient an effective amount of a novel peptidedescribed herein. Cluster headache is a condition that involves, as itsmost prominent feature, recurrent, severe headaches on one side of thehead, typically around the eye (see Nesbitt et al., BMJ, Vol. 344:e2407,2012). Some doctors and scientists have described the pain resultingfrom cluster headaches as the most intense pain a human can endure—worsethan giving birth, burns or broken bones. Cluster headaches often occurperiodically: spontaneous remissions interrupt active periods of pain.Cluster headaches are often accompanied by cranial autonomic symptoms,such as tearing, nasal congestion, ptosis, pupil constriction, facialblushing, sweating, and swelling around the eye, often confined to theside of the head with the pain. The average age of onset of clusterheadache is ˜30-50 years. It is more prevalent in males with a male tofemale ratio of about 2.5:1 to about 3.5:1. Sphenopalatine ganglion(SPG) stimulation has been used for the treatment of cluster headache. Aneurostimulation system, which delivers low-level (but high frequency,physiologic-blocking) electrical stimulation to the SPG, hasdemonstrated efficacy in relieving the acute debilitating pain ofcluster headache in a recent clinical trial (see Schoenen J, et al.,Cephalalgia, Vol. 33(10):816-30, 2013). In view of this evidence andbecause PACAP is one of the major neurotransmitters in SPG, inhibitionof PACAP signaling with a PAC1 antagonist described herein is expectedto have efficacy in treating cluster headache in humans.

Other conditions associated with PACAP biological activity that may betreated according to the methods of the invention include, but are notlimited to, inflammatory skin conditions, such as rosacea (see U.S.Patent Publication No. 20110229423), chronic pain syndromes, such asneuropathic pain (see Jongsma et al., Neuroreport, Vol. 12: 2215-2219,2001; Hashimoto et al., Annals of the New York Academy of Sciences, Vol.1070: 75-89, 2006), tension-type headaches, hemiplegic migraine, retinalmigraine, anxiety disorders, such as posttraumatic stress disorder (seeHammack and May, Biol. Psychiatry, Vol. 78(3):167-177, 2015), irritablebowel syndrome, and vasomotor symptoms (e.g. hot flashes, facialflushing, sweating, and night sweats), such as those associated withmenopause. In one embodiment, the condition is chronic pain. In anotherembodiment, the condition is neuropathic pain. In any of the methodsdescribed herein, the treatment can comprise prophylactic treatment.Prophylactic treatment refers to treatment designed to be taken beforethe onset of a condition or an attack (e.g. before a migraine attack oronset of a cluster headache episode) to reduce the frequency, severity,and/or length of the symptoms (e.g. migraine or cluster headaches) inthe patient.

The following examples, including the experiments conducted and theresults achieved, are provided for illustrative purposes only and arenot to be construed as limiting the scope of the appended claims.

Example 1 General Peptide Synthesis

The following general procedure was followed to synthesize peptides ofthe invention. Peptide synthesis was carried our using N^(α)-Fmocsolid-phase peptide synthesis (SPPS) methodologies with appropriateorthogonal protection and resin linker strategies.

The following materials have been used. N^(α)-Fmoc protected amino acidswere purchased from Advanced ChemTech (Louisville, Ky.), MidwestBIO-TECH (Fishers, Ind.), Chem-Impex International (Wood Dale, Ill.),Novabiochem (San Diego, Calif.), Protein Technologies (Tucson, Ariz.),Combi-Blocks (San Diego, Calif.), Chem-Impex International (Wood Dale,Ill.), Bachem (Torrance, Calif.), or GL Biochem (Shanghai, China). AG1-X2 ion-exchange resin (200-400, acetate) was purchased from Chem-ImpexInternational. Rink Amide MBHA resin was purchased from PeptidesInternational (100-200 mesh, 1% DVB, RFR-1063-PI, 0.52 mequiv/g initialloading, Peptides International, Louisville, Ky.). SP Sepharosehigh-performance resin was purchased from GE Healthcare Life Sciences.The following chemicals were purchased and used directly without furtherpurifications: N,N-diisopropylethylamine (DIEA), 2,2,2-trifluoroethanol,trifluoroacetic acid (TFA), acetic acid, acetic anhydride, formic acid,piperidine, 4-methyl-piperidine, N,N′-diisopropylcarbodiimide (DIC),6-chloro-1-hydroxybenzotriazole (6-Cl-HOBt), ethylcyano(hydroxyimino)acetate (Oxyma), 3,6-dioxa-1,8-octanedithiol (DODT),triisopropylsilane (TIS), cysteine, cystine (Sigma-Aldrich, Milwaukee,Wis.); dichloromethane (DCM, Mallinckrodt Baker, Inc.);N,N-dimethylforamide (DMF, Fisher Scientific); HPLC-quality water andacetonitrile (Burdick and Jackson); 1.0 M Tris-HCl, pH=8.0 (Teknova).

The following side chain protection strategies were employed forstandard amino acid residues: Asn(Trt), Asp(O^(t)Bu), Arg(Pbf),Cys(Trt), Gln(Trt), Glu(O^(t)Bu), His(Trt), Lys(N-Boc), Orn(N-Boc);Ser(O^(t)Bu), Thr(O^(t)Bu), Trp(Boc), and Tyr(O^(t)Bu). The peptidesdescribed could be synthesized at various temperatures using variousautomatic peptide synthesizers.

Peptide syntheses were carried out by solid phase peptide synthesis(SPPS). Amino acid couplings were conducted using a variety of automatedsynthesizers: room temperature coupling with Intavis Multipep Rsi or CSBio 336X and higher temperature couplings with Tribute or CEM Liberty.After completion of the synthese, peptides were deprotected and cleavedfrom the resin. For some analogs, the N-termini of the peptides weremodified (such as by N-acylation) after deprotection and cleavage fromresin. Peptides were oxidatively folded and purified by reverse phasechromatography. In some cases, salt exchange was performed usingion-exchange resin prior to testing.

The following non-limiting examples illustrate the application ofdifferent coupling methods which could be used to synthesize thepeptides of the invention.

Coupling method 1—Room temperature coupling of peptides using automatedpeptide synthesizer Intavis Multipep Rsi (INTAVIS BioanalyticalInstruments AG, Cologne, Germany) was conducted as described in detailbelow. Dry resin (0.012 mmol, per well) was added to a Phenomenex deepwell protein precipitation plate (CEO-7565, 38710-1) using a resinloader (Radley). Amino acids (5 mol equiv, 0.5 M in DMF) werepreactivated (1 min) with Oxyma (5 mol equiv, 0.4 M in DMF) and DIC (7.5mol equiv, 1.0 M in DMF). Preactivated amino acids were transferred tothe appropriate well. Resins were incubated for 30 min and drained, andthe cycle was repeated. Following the second amino acid incubation, theplates were drained and washed with DMF multiple times. The Fmoc groupswere then removed by two incubation sequences of a 20% piperidine in DMFsolution. The resin was drained and washed with DMF 10 times (4 mL percolumn of 8 wells). After removal of the final Fmoc protecting group,the resin was washed with DCM multiple times and allowed to air-dry.

Coupling method 2—Room temperature couping of peptides using automatedpeptide synthesizer CS Bio 336X single-channel solid phase peptidesynthesizer (Menlo Park, Calif.) was conducted as following. Dry resin(0.2 mmol Rink amide or pre-loaded acid Wang resin) was weighed into aCS Bio reaction vessel. The reaction vessel was connected onto areaction vessel holder in the reaction chamber. The resin was swollen inDMF (˜10 mL) for 15 min. Fmoc-amino acid (1.0 mmol, Midwest Biotech orNovabiochem) was dissolved in 2.5 mL of 6-Cl-HOBt (1.0 M in DMF). Tothis solution was added 1.0 mL DIC (1.0 M in DMF) and the overallmixture was agitated with nitrogen bubbling for 15 min to accomplishpre-activation and then transferred onto the resin. The mixture wasshaken for 2 h and the resin was filtered and washed (3×DMF, 2×DCM, and3×DMF subsequently). Fmoc-removal was accomplished by treatment with 20%piperidine (or 4-methylpiridine) in DMF (5 mL, 2×15 min). The resin wasfiltered and washed (3×DMF). All residues were single coupled throughrepetition of the Fmoc-amino acid coupling and Fmoc removal stepsdescribed above.

Coupling method 3—Higher temperature coupling via Infrared (IR) heatingusing automated peptide synthesizer Tribute (Protein Technologies, Inc.,Tucson, Ariz.) was conducted as described herein. Dry resin (0.3 mmol)was loaded onto a reaction vessel and then wetted with 30% DMF in DCM(˜5 mL) and allowed to swell for 30 min. Fmoc amino acids (4-5 equiv.)were preactivated with 6-Cl-HOBt (or Oxyma) and DIC (4 equiv.). Thepreactivated solution is added to the resin and vortexted for 5 min at75° C. The Fmoc deprotection step was accelerated in a similar manner at50° C. in a period of 4×30 sec.

Coupling method 4—Higher temperature coupling (0.2 mmol) via microwaveheating using automated peptide synthesizer CEM Liberty Blue (CEMCorporation, Matthews, N.C.) was conducted as follows. Similardeprotection and coupling reagents as described above could be used inthe synthesis with minor modifications to accommodate the specificationsof the synthesizer. Typical reaction coupling and deprotectionconditions can occur temperatures of 50° C., 75° C., or 90° C.,depending on the amino acid used. Amino acid coupling reactions wereperformed with 5 fold excess of Fmoc-amino acids and in the presence ofDIC (1.0 M in DMF) and Oxyma (0.5 M in DMF, with 0.05 M DIEA). The Fmocgroups were removed by excess of 20% piperidine (or 4-methylpiperidine)in DMF several times at 90° C.

N-acylation of peptides: After removal of the final Fmoc protectinggroup, the peptide-resin (0.2 mmol) was transferred to a solid phaseextraction (SPE) filter tube and washed with DMF (×3), DCM (×3), andallowed to dry under vacuum. The resin was treated with excess of aceticanhydride (10% in DMF, 10 mL×2), followed by washed with DMF (×3) toproduce the N-acylated peptides.

Deprotection and cleavage from resin after coupling method 1: To thebottom of the filter plate was affixed a drain port sealing mat(ArcticWhite, AWSM-1003DP). To the resin (0.012 mmol) in each well wasadded triisopropylsilane (100 μL), DODT (100 μL), and water (100 μL)using a multichannel pipet. To the resin in each well was added TFA (1.0mL) using a Dispensette Organic dispenser. The top of the plate wascovered with another drain port sealing mat. The mixture was agitated ona plate shaker for 2 h. The top mat was removed, and the bottom mat wasremoved and the plate was allowed to drain into a solid bottom 96-wellplate, aided by vacuum. This plate was evaporated on the genevac for 8h. To each well of this 96-well plate was added 1.8 mL of cold diethylether using a Dispensette Organic dispenser and precipitates formed andeach mixture was transferred into a new 96-well filter place using amultichannel pipet with wide bore tips and allowed to drain. The crudepeptides in the plate were air-dried for 1 h and the bottom of thefilter pate was capped. To this plate 1 ml of 50/50 acetonitrile/waterwas added to each well and the plate was vortex overnight. Additional 1ml of water was added to each well and with a pipette the wells weremixed. The resulting solutions (˜2.0 mL total per well) were thenfiltered into a new 96-well plate and set aside.

Deprotection and cleavage from resin after coupling method 2-4: Afterremoval of the final Fmoc protecting group, the peptide-resin (0.2 mmol)was transferred to a 25 mL solid phase extraction (SPE) filter tube andwashed with DMF (×3), DCM (×3), and allowed to dry under vacuum. Thepeptides were cleaved from resin with 1 mL of TIS, 1 mL of DODT, 1 mL ofH₂O, and 20 mL of TFA and the resulting suspension was gently agitatedat room temperature for 3.5 h. The resin was filtered and washed withadditional TFA (10 mL) and the overall solution was concentrated underreduced pressure until the final volume ˜10 mL. The remaining residuewas precipitated with cold cyclopropylmethyl ether (50 mL). Theresulting white suspension was filtered, washed with coldtert-butylmethyl ether (25 mL), and the resulting white suspension wasfiltered again. The collected material was dried in a desiccator underreduced pressure overnight to afford crude linear peptide as anoff-white solid.

Peptide oxidative folding of cleaved peptides from coupling method 1:The oxidative folding of the 96 peptide array was performed in paralleland at high dilution using an array of 50 mL centrifuge tubes in thefollowing manner. In a 4 L bottle added 175 mL of acetonitrile, 3125 mLof water, 350 mL of pH 8 Tris-HCl, 23.45 mL of 0.1 M cystine, and 3.5 mL1.0 M cysteine to form the stock folding solution. To each tube 35 mL ofthe above folding solution was added, followed by the addition of thedissolved peptide solution using the Tecan automated liquid handler andthe resulting folding reactions were allowed to stand at roomtemperature overnight. To each tube in the array was added 1 mL ofglacial acetic acid to quench the reaction. A 96-well ion-exchangefilter plate was prepared by pipetting 1.0 mL SP Sepharose HighPerformance resin (slurry in 0.2 M sodium acetate/20% ethanol, GEBiosciences) and 2.0 mL of sodium acetate (×3, 20 mM, pH=4) to each welland then was allowed to drain slowly. To this plate the peptide foldingsolutions in the 50 mL centrifuge tubes prepared above were added usingthe Tecan liquid handler. The resin was washed (2.0 mL, 20 mM sodiumacetate, pH=4.0), and the peptide was eluted off of the resin manuallyon a vacuum manifold with 2.0 mL (1.0 M sodium chloride, 20 mM sodiumacetate, pH=4) into a solid bottom 96-well plate.

Peptide oxidative folding of cleaved peptides from coupling method 2-4:To a dried PAC1 peptide crude (typically 0.8˜1.2 g dry weight from a 0.2mmol reaction) was added 40˜60 mL of 50:50 water/acetonitrile and themixture was vortexed and sonicated until a clear solution was obtained.The overall solution was filtered and set aside. In a separated 1-Lbottle a folding buffer was prepared by mixing 850 mL of DI water, 48 mLof acetonitrile, 95 mL of 1.0 M Tris-HCl (pH=8.0), 6.4 mL of 0.15-0.17 Mcystine aqueous solution, and 0.96 mL 1.0 M aqueous solution ofcysteine. The crude PAC1 peptide solution prepared above was pouredslowly into the folding buffer for a final peptide concentration of 0.3mM and was allowed to stand at room temperature overnight. The foldingmixture was quenched with excess amount of acetic acid (25 mL) andfiltered (0.45 m polyethylene) to yield a colorless solution.

Alternatively, folding can be conducted at higher final concentrationthan described above. Crude peptide was first solubilized in 90:10water/acetonitrile solution at ˜5.6 mg/mL. Cysteine (1.0 M, 0.6 mL) andCystine (0.17 M, 4 mL) solutions were then added for a finalconcentration of 0.1 M each and the resulting solution was mixedthoroughly. Lastly, 1 M Tris HCl (pH 8.0, 60 mL) solution was added toachieve final peptide concentration of 1.4 mM. The folding mixture wasincubated overnight at room temperature and quenched with TFA to ˜ pH3.0. The mixture was then filtered with a 0.45 u filter unit.

Purification: The filtered folded peptide solutions from above werepurified by mass-triggered preparative HPLC. The collected fractionswere pooled and lyophilized. Final QC and peptide quantification wasperformed by chemiluminescent nitrogen detection (CLND) as mentionedabove. Peptides with >90% purity and correct m/z ratio were submittedfor assay.

Trifluoroacetate-acetate salt exchange: Following lyophilization, thePAC1 peptide trifluoroacetate salt in a 50-mL Falcon tube wasredissolved in DI water (100-150 □L/1 mg peptide) and set aside. Toanother Falcon tube was added wet AG 1-X² ion-exchange resin (25 mg/per1 mg peptide) washed three times with 1.6 N acetic acid and three timeswith 0.16 N acetic acid (0.6˜1.0 mL per 1 mg dry peptide). The peptidesolution was poured into the prewashed resin (additional water was usedto transfer the residual peptide, total ˜200 □L DI water per 1 mg drypeptide). The Falcon tube was capped and placed into a tube holder on anEppendorf ThermoMix and agitated (400 rmp) at 20° C. overnight. Theresulting suspension was filtered and the resin was washed with more DIwater (×3, 200 □L DI water per 1 mg peptide). The combined aqueousphases were frozen and lyophilized to give the desired peptide acetatesalt as a white solid.

Example 2 PAC1 Peptide Antagonist Functional Screen

In order to determine the potency of peptide antagonists at the PAC1receptor, functional cAMP accumulation assays using LANCE Ultra cAMPassay kit to measure the agonist and antagonist activity of PAC1peptides were used. SHSY-5Y (human neuroblastoma cell line) and RG-2(rat glioma cell line) cells endogenously express human and rat PAC1receptors, respectively. Agonist activity of PAC1 peptides was tested byincubating with SHSY-5Y at 2,000 cells per well or RG-2 at 5,000 cellsper well for 15 minutes at room temperature. PAC1 receptor agonistPACAP38 or maxadilan was included as a positive control. The reactionwas stopped by adding detection mixture, europium (Eu) chelate-labeledcAMP tracer and cAMP-specific monoclonal antibodies labeled with theULight™ dye, to all wells followed by a 60-minute incubation at roomtemperature. The assay plates were then read on an EnVision instrumentat an emission wavelength of 665 and 615 nm and data were analyzed usingGenedata Screener or GraphPad Prism software. A similar procedure wasused in the antagonist assays by using a fixed concentration of PAC1agonist. The concentrations of agonists were chosen based on their EC₈₀values at PAC1 receptors in SHSY-5Y or RG-2 cells. SHSY-5Y or RG-2 cellswere added into 96-well half-area white plates. Serially diluted PAC1peptides at ten concentrations were added and incubated for 30 minutesat room temperature. PAC1 agonist at EC₈₀ concentration was then addedand further incubated for 15 minutes at room temperature. The reactionwas stopped by adding detection mixture to all wells followed by a60-minute incubation at room temperature. The assay plates were read andanalyzed the same way as that of agonist assay. Cells, agonists, andPAC1 peptides were prepared in freshly made assay buffer (F-12, 0.5 mMIBMX, 0.1 or 0.4% BSA (pH 7.4)). Detection mixture was made by mixing a1/50 dilution of the Eu-cAMP tracer stock solution in cAMP DetectionBuffer and a 1/150 dilution of the ULight-anti-cAMP stock solution incAMP Detection Buffer. Europium (Eu) chelate-labeled cAMP tracer,cAMP-specific monoclonal antibodies labeled with the ULight™ dye, BSA,and detection buffer were provided in LANCE Ultra cAMP Kit fromPerkinElmer.

TABLE 3 In vitro data IC₅₀: A ≥ 100 nM; 100 nM > B ≥ 1 nM; 1 nM > C ≥0.01 nM; 0.01 nM > D hPAC1 rPAC1  Calc  IC50 with IC50 with sequenceAvg. PACAP38 maxadilan ID# MW MS observed challenge challenge 1 4851.55971.2; 1213.7; 1617.9 C D 2 4855.55 972.0; 1214.7; 1619.2 C C 3 4543.22909.5; 1036.6; 1515.1 C C 5 4062.75 1016.7; 1355.1 B B 6 4423.94 885.9;1106.9; 1475.8 C C 7 4903.43 981.6; 1226.8; 1635.5 C C 8 4840.53 969.0;1210.9; 1614.3 C C 9 4838.5 968.7; 1210.4; 1613.6 C C 10 4833.5 967.6;1209.2; 1611.8 C C 11 4870.56 975.0; 1218.4; 1624.3 C C 12 4665.39934.0; 1167.2; 1555.9 C D 13 4635.36 928.0; 1159.7; 1545.9 C C 144633.32 927.5; 1159.2; 1545.3 C C 15 4574.24 1144.4; 1525.4 C C 164395.02 1099.6; 1465.8 C C 17 4679.37 1170.6; 1560.4 C D 18 4693.441174.2; 1565.3 C D 19 4450.14 890.8; 1113.3; 1483.9 B C 20 4277.911070.2; 1246.8 C C 21 4421.05 1105.9; 1474.3 C C 22 4870.56 975.1;1218.4; 1624.0 C C 23 4898.61 980.6; 1225.4; 1633.6 C D 24 4872.531218.9; 1624.9 C C 25 4870.56 975.0; 1218.4; 1624.2 C D 26 4461.12893.2; 1116.1; 1487.8 C C 27 4515.17 904.0; 1129.5; 1505.7 C C 284489.13 898.7; 1123.1; 1497.1 C C 29 4448.12 890.5; 1112.8; 1483.4 C C30 4474.15 895.5; 1119.4; 1492.2 C C 31 4667.36 934.3; 1167.7; 1556.5 CD 32 4732.43 947.3; 1183.9; 1578.2 C C 33 4675.34 936.0; 1169.7; 1559.2B C 34 4746.46 950.0; 1087.4; 1582.8 C C 35 4476.13 896.2; 1119.8;1492.8 C C 36 4450.1 890.8; 1113.3; 1484.1 C C 37 4310.98 1078.6; 1437.7B C 39 4872.51 975.4; 1218.9; 1624.8 C C 40 4812.42 963.5; 1203.9;1604.9 B B 41 4838.5 968.6; 1210.4; 1613.7 C C 42 4685.37 938.0; 1172.1;1562.4 C C 43 4813.55 963.5; 1204.1; 1605.3 C D 44 4841.56 969.1;1211.2; 1614.6 C D 45 4822.51 965.3; 1206.3; 1608.2 C C 46 4875.89975.9; 1219.7; 1626.1 B C 47 4881.58 977.1; 1221.2; 1627.9 C C 484843.57 969.5; 1211.7; 1615.2 C C 51 5142.85 1029.4; 1286.5; 1715.1 B B52 5104.81 1021.7; 1277.0; 1702.3 B C 53 5047.76 1010.4; 1262.7; 1683.3B C 54 4861.59 973.2; 1216.2; 1621.3 B C 55 4911.61 1228.8; 1637.9 B C56 4815.52 963.8; 1204.7; 1605.8 C C 57 4789.48 958.8; 1198.2; 1597.2 CC 58 4872.57 975.3; 1218.8; 1624.9 C C 59 4846.54 970.2; 1212.4; 1616.2C C 60 4894.62 979.9; 1224.4; 1632.3 C C 61 4543.22 909.5; 1136.6;1515.1 C C 62 5009.71 1002.8; 1253.2; B C 1670.6 63 4405.05 881.8;1102.0; 1469.0 C C 64 4872.57 975.3; 1218.9; 1624.7 B C 65 4377.04876.3; 1095.0; 1459.7 C C 66 5058.78 1012.5; 1265.5; C C 1686.9 674932.63 987.3; 1233.9; 1645.1 B C 68 4808.53 962.5; 1202.9; 1603.6 C C69 4950.69 990.8; 1238.4; 1651.0 C C 70 4969.73 994.8; 1243.2; 1657.2 BB 71 4983.68 997.5; 1246.7; 1661.9 C C 72 4681.34 937.2; 1171.2; 1561.3C C 73 4775.58 955.2; 1193.8; 1591.4 C C 74 4807.54 962.3; 1202.8;1603.3 C C 75 4517.19 904.3; 1130.1; 1506.4 C D 76 4736.51 948.3;1185.1; 1579.8 C C 77 4910.62 983.0; 1228.4; 1637.6 C C 78 4828.56966.3; 1207.6; 1609.8 C C 79 4746.46 950.0; 1187.4; 1582.8 C D 804766.49 954.2; 1192.4; 1589.3 C D 81 4763.4 953.5, 1191.7, 1588.6 B C 824747.45 950.2, 1187.7, 1583.3 C C 83 4722.43 1181.4, 1574.9 C C 844910.62 982.9, 1228.4, 1637.7 C C 85 4755.55 945.0; 1180.8; 1574.3 C C86 4910.62 983.0, 1228.4, 1637.7 C C 87 4804.5 961.8, 1201.9, 1602.3 C C88 4941.64 989.2, 1236.2, 1647.9 C C 89 4779.49 956.6, 1195.7, 1593.8 BC 90 4722.5 1181.5, 1574.8 C C 91 4704.46 1176.9, 1568.8 C C 92 4803.621201.7, 1601.8 C C 93 4661.33 1166.1, 1554.5 B B 94 4817.54 1205.2,1606.6 C C 95 4838.56 968.5, 1210.4, 1613.7 B B 96 4780.47 957.0,1196.0, 1594.3 B C 97 4809.52 962.8, 1203.3, 1603.8 B B 98 4736.46948.2, 1185.0, 1579.6 B C 99 4827.54 966.5, 1207.6, 1609.9 C C 1004675.36 935.8, 1169.6, 1559.2 B B 101 4661.33 933.1, 1166.1, 1554.4 B B102 4679.37 936.5, 1170.6, 1560.5 B C 103 4693.4 939.5, 1174.1, 1565.2 BC 104 4855.58 971.9, 1214.7, 1619.3 C C 105 4895.57 979.8, 1224.7,1632.6 C C 106 4923.62 985.5, 1231.7, 1641.8 C C 107 4755.55 952.0;1189.7; 1585.8 C C 108 4961.66 1241.2; 1654.6 C C 109 4938.67 1235.4;1646.8 B C 110 4988.69 1247.9; 1663.7 C C 111 4941.74 1236.2, 1648.4 C C112 4941.74 1236.2; 1648.4 C C 113 5009.71 1002.8; 1253.2; 1670.6 B C114 4800.56 960.9; 1200.9; 1601.1 C C 115 4720.46 945.0, 1180.8, 1574.3C C 116 4830.59 1208.4, 1610.9 C C 117 4816.55 1204.9, 1606.3 C C 1184731.53 1183.7, 1577.9 C C 119 4759.5 952.7, 1190.6, 1587.3 C C 1204805.57 961.8, 1202.2, 1602.6 C C 121 4839.61 968.7, 1210.7, 1613.8 C C122 4837.63 968.3, 1210.1, 1613.3 C C 123 4704.46 941.8, 1176.9, 1568.9C C 124 4820.51 964.8, 1205.8, 1607.6 C C 125 4806.56 962.2, 1202.4,1602.8 A B 126 4787.52 958.3, 1197.7, 1596.6 C C 127 4769.45 954.7,1193.2, 1590.4 C C 128 4825.56 966.2, 1207.1, 1609.3 C C 129 4755.551189.7, 1585.9 C C 130 4771.55 1193.8, 1591.2 C D 131 4780.58 1195.9,1594.1 C C 132 4746.56 950.3, 1187.4, 1583.0 C D 133 4782.55 1196.4,1594.8 C C 134 4786.52 1197.4, 1596.1 C C 135 4823.54 1206.8, 1608.7 C C136 4690.43 1173.3, 1564.2 C C 137 4706.43 942.1, 1177.4, 1569.5 C C 1384800.55 1200.9; 1600.9 C C 139 4850.56 971.0, 1213.4, 1617.6 C C 1404720.51 944.9; 1180.9; 1574.3 C C 141 4802.52 961.3; 12014; 1601.4 1424676.35 936.2, 1169.8, 1559.6 B B 143 4690.37 939.0, 1173.4, 1564.1 C C144 4788.45 958.5, 1197.8, 1596.8 C C 145 4895.61 979.9, 1224.8, 1632.5C C 146 4738.44 948.4, 1185.3, 1580.1 C C 147 4819.61 1205.7, 1607.3 C C148 4805.57 1202.1, 1602.6 C C 149 4871.58 975.2, 1218.6, 1624.5 C C 1504846.59 1212.4, 1616.3 C C 151 4856.53 972.1, 1214.9, 1619.4 C C 1524828.52 1207.9, 1610.2 C C 153 4789.48 958.8, 1198.2, 1597.2 B C 1544934.56 987.8, 1234.4, 1645.6 C C 155 4861.51 973.1, 1216.2, 1621.2 B C156 4884.59 977.7, 1221.8, 1629.0 C C 157 4867.55 1217.7, 1623.2 B C 1584819.47 964.8, 1205.6, 1607.2 C C 159 4820.46 964.8, 1205.9, 1607.3 B C160 5532.23 1107.3, 1383.8, B C 1844.8 161 4908.65 982.5, 1228.0, 1637.1C C 162 4819.51 964.6, 1205.7, 1607.3 B C 163 4762.42 953.3, 1191.3,1588.2 C C 164 4872.57 975.3, 1218.9, 1624.9 C 166 4818.53 964.5,1205.4, 1606.8 C 167 4900.54 980.9, 1225.9, 1634.3 C C 168 4723.38945.5, 1181.7, 1575.3 C C 169 4695.33 939.9, 1174.6, 1565.8 C C 1704865.58 974.0, 1217.2, 1622.4 C 171 4846.54 970.1, 1212.4, 1616.2 C 1724856.57 972.2, 1215.0, 1619.6 C 173 4838.51 968.5, 1210.4, 1613.5 C 1744837.53 968.3, 1210.1, 1613.2 B C 175 4780.47 957.0, 1195.9, 1594.3 C176 4851.56 971.2, 1213.6, 1617.9 B 177 4967.63 994.3, 1242.7, 1656.7 C178 4268.9 854.7, 1068.0, 1423.6 C C 179 4679.41 936.7, 1170.6, 1560.4 CC 180 4651.36 931.1, 1163.7, 1551.2 C C 181 4765.46 954.0, 1192.1,1589.2 B C 182 4837.57 968.3, 1210.2, 1613.3 C 183 4795.49 959.9,1199.7, 1599.3 B C 184 4779.49 956.8, 1195.6, 1593.8 C 185 4852.54971.4, 1213.9, 1618.3 C 186 4737.41 948.3, 1185.2, 1579.8 C 187 4848.331212.8 C 188 4720.42 944.8, 1180.9, 1574.1 C 189 4836.54 968.2, 1209.9,1612.9 C 190 4709.4 942.7, 1178.2, 1570.4 C 191 4752.42 951.3, 1188.9,1585.2 C 192 4737.45 948.3, 1185.2, 1579.8 C 193 4779.49 956.7, 1195.7,1593.9 C 194 4739.43 948.8, 1185.7, 1580.6 C 197 5251.04 1050.9, 1313.5,C 1751.0 198 4720.47 945.1, 1180.9, 1574.2 C 199 5526.29 1106.0, 1382.4,C 1842.8 200 4750.45 951.0, 1188.4, 1584.2 C 201 4636.3 928.2, 1159.9,1546.3 C 202 4766.45 954.2, 1192.4, 1589.3 C 203 4736.46 948.1, 1184.9,1579.3 C 204 5663.43 1133.6, 1416.8, C 1888.6 205 4765.46 953.9, 1192.2,1589.3 C 206 4780.47 956.9, 1195.9, 1594.3 C 207 4724.41 945.6, 1181.9,1575.3 C 208 4749.46 950.8, 1188.3, 1583.8 C 209 4692.41 939.4, 1173.9,1564.9 C 210 4738.44 948.7, 1185.4, 1580.5 B B 211 4736.46 948.0,1184.9, 1579.4 B C 213 4718.36 944.5, 1180.4, 1573.6 B C 214 4651.36931.1, 1163.3, 1551.2 B C 215 4736.46 948.3, 1184.9, 1579.6 B C 2164623.31 925.5, 1156.6, 1541.8 B C 217 4109.74 822.8, 1028.3, 1370.8 B B218 4195.83 1049.8, 1399.6 B B 219 4140.71 829.0, 1036.0, 1381.1 B B 2204254.81 851.8, 1064.6, 1418.9 B B 221 4239.84 848.9, 1060.8, 1413.9 B B222 4272.85 855.5, 1069.1, 1425.2 D B 223 4257.88 852.5, 1065.3, 1420.1D B 224 4285.89 858.1, 1072.3, 1429.4 C B 225 4270.92 855.1, 1068.5,1424.4 C B 226 4750.49 951.1, 1188.4, 1584.3 C C 227 4678.39 936.5,1170.4, 1560.1 C C 228 4779.49 956.8, 1195.7, 1593.9 C C 229 4737.41948.3, 1185.2, 1579.9 C C 230 4708.41 942.6, 1177.9, 1570.2 C C 2314761.47 1191.3 C C 232 1785.23 595.9, 893.6 A B 233 4708.41 942.5,1177.9, 1570.3 B C 234 4610.27 922.9, 1153.3, 1537.3 C C 235 4748.47950.7, 1187.9, 1583.6 C C 236 4751.43 951.1, 1188.7, 1584.5 C C 2374635.36 927.8, 1159.6, 1545.8 B C 238 4622.32 925.5, 1156.4, 1541.4 C C239 4652.3 931.5, 1163.8, 1551.4 B C 240 4114.74 823.9, 1029.6, 1372.4 BC 241 4223.84 1056.8, 1408.7 B B 242 4208.87 842.8, 1053.1, 1403.7 B B243 4127.78 826.4, 1032.8, 1376.7 B B 244 4228.84 846.8, 1058.1, 1410.4B C 245 4213.87 843.6, 1054.2, 1405.4 B C 246 4322.97 865.5, 1081.6,1441.8 B B 247 4241.88 849.3, 1061.3, 1414.7 B B 248 4634.31 927.7,1159.4, 1545.6 B C 249 4707.42 942.3, 1177.7, 1569.8 B C 250 4665.39934.0, 1167.2, 1555.8 B C 251 4660.34 933.0, 1165.9, 1554.3 B C 2524592.23 919.3, 1148.8, 1531.4 B C 253 4764.47 953.8, 1191.9, 1588.8 C C254 4709.4 942.8, 1178.1, 1570.4 C C 255 4691.36 939.2, 1173.6, 1564.4 BC 256 4341.01 1086 B B 257 4464.1 1116.8 B C 258 4578.2 916.5, 1145.3,1527.1 B C 259 4563.23 913.5, 1141.6, 1521.8 B C 260 4618.31 924.5,1155.4, 1540.2 B C 261 4677.33 936.3, 1170.1, 1559.7 B C 262 4625.32926.0, 1157.2, 1542.6 B C 263 4704.4 941.7, 1176.9, 1568.8 B C 2644576.27 916.1, 1144.8, 1526.3 B C 265 4665.34 934.0, 1167.2, 1555.9 A B266 4721.34 945.0, 1181.2, 1574.6 B C 267 4591.24 919.0, 1148.6, 1531.1B C 268 4690.37 938.9, 1173.4, 1564.1 B B 269 4620.28 924.8, 1155.8,1540.9 B B 270 4596.24 920.0, 1149.8, 1532.8 B C 271 4581.27 917.1,1146.1, 1527.9 B C 272 4824.48 965.7, 1206.9, 1608.9 A B 273 4881.54977.0, 1221.2, 1628.1 A B 274 4096.7 820.2, 1025.0, 1366.3 B B 2754655.31 931.8, 1164.6, 1552.6 B B 276 4752.38 951.4, 1188.9, 1585.0 A B277 4738.39 948.5, 1185.3, 1580.1 B B 278 4752.42 951.3, 1188.9, 1585.0A B 279 4695.33 939.8, 1174.7, 1565.8 B B 280 4681.3 937.2, 1171.1,1561.3 A 281 4752.42 951.3, 1188.9, 1584.8 B 282 4770.48 954.5, 1192.3 AB 283 4722.44 945.3, 1181.4, 1574.9 B C 284 4752.38 951.3, 1188.9, A B285 4681.3 937.1, 1171.2, 1561.3 A 286 4709.4 942.7, 1178.2, 1570.5 B B287 4694.39 939.7, 1174.3, 1565.6 B B 288 4780.43 1195.8, 1594.3 B B 2894700.39 1175.9 B B 290 5051.74 1263.6 B B 291 4766.45 1192.4 A 2924766.45 1192.4 A B 293 4638.32 1160.3 B C 294 5008.72 1002.5, 1253.0, BC 1670.3 295 4739.38 948.7, 1185.6, 1580.4 B C 296 4708.41 1177.9 B B297 4766.45 954.2, 1192.4, 1589.7 B B 298 4734.38 1184.4 B B 299 4738.39948.5, 1185.4, 1580.4 B B 300 5018.67 1004.6, 1255.5, B B 1673.8 3014791.46 959.1, 1198.7, 1597.8 B B 302 4761.39 1191.2 B B 303 4752.38951.8, 1188.8, 1584.8 B B 304 4752.42 951.3, 1188.8, 1584.9 B B 3064778.46 956.7, 1195.4, 1593.6 B B 307 4752.38 951.3, 1188.8, 1584.7 A B308 4695.33 940.0, 1174.6, 1565.7 A B 309 4752.42 951.3, 1188.9, 1584.9A B 310 4753.36 951.5, 1189.2, 1585.3 A B 311 4695.37 940.0, 1174.7,1565.8 A B 312 5023.73 1005.6, 1256.8, A A 1675.2 313 4753.41 951.6,1189.2, 1585.3 A A 314 5035.74 1007.8, 1259.4, A B 1679.1 315 5048.741010.8, 1263.1, B B 1684.1 316 5010.65 1003.0, 1253.4, B B 1670.8 3175009.66 1002.8, 1253.3, B B 1670.5 318 5009.71 1003.0, 1253.3, B B1670.8 319 5025.71 1006.0, 1257.3, B B 1675.8 320 5018.67 1004.5,1255.4, A B 1673.5 321 5009.66 1002.8, 1253.2, A B 1670.5 322 5009.711002.6, 1253.3, B B 1670.7 323 4995.68 1000.0, 1249.7, B B 1665.7 3244978.69 996.7, 1245.7, 1660.3 A A 325 5010.69 1003.0, 1253.6, B B 1670.8326 4952.61 991.5, 1238.9, 1651.4 B 327 4982.68 997.3, 1246.5, 1661.6 BB 328 5050.76 1010.9, 1263.7, B 1684.8 329 5009.71 A 330 5048.74 B 3315025.71 B 332 5023.73 1005.5, 1256.7, B 1675.3 333 5032.74 1007.3,1259.0, B 1678.3 334 5023.73 1005.5, 1256.8, B 1675.6 335 4991.65 999.2,1248.7, 1664.6 A 336 5010.65 1002.5, 1253.2, A 337 4979.72 996.7,1245.7, 1660.4 B 338 4981.65 997.2, 1246.2, 1661.2 A 339 5009.71 1002.7,1253.2, B B 1670.6 340 5010.65 A 341 5010.65 A 342 5007.63 A 343 5023.73B 344 4952.61 B 345 4966.68 B 346 5009.71 1002.7, 1253.2, A 1670.5 3475023.73 1005.5, 1256.8, B 1675.3 348 5038.75 1008.5, 1260.4, B 1680.3349 4996.67 1000.2, 1249.9, B 1665.9 350 5009.66 1002.8, 1253.2, B1670.7 351 5024.68 1005.8, 1256.9, B 1675.8 352 5037.72 1008.4, 1260.3,B 1679.9 353 5038.75 1008.5, 1260.4, B 1680.3 354 4996.67 1000.2,1249.8, B 1666.3 355 5035.74 1008.0, 1259.7, B 1679.5 356 5035.741007.8, 1259.8, B 1679.2 357 5021.76 1005.0, 1256.2, B 1674.6 3585011.68 1003.1, 1253.7, B 1671.2 359 5011.68 1003.3, 1253.6, B 1671.6360 5051.79 1011.0, 1263.7, B 1684.7 361 5051.79 1011.3, 1263.8, A1684.5 362 4862.53 973.3, 1216.4, 1621.4 A 363 4910.58 982.8, 1228.3,1637.3 B 364 4895.6 980.0, 1224.7, 1632.6 B 365 4952.66 1238.9 B 3664983.67 997.5, 1246.7, 1661.9 B 367 5025.71 1005.9, 1257.2, B 1675.8 3685023.73 1005.7, 1256.6, B 1675.2 369 5023.73 1005.5, 1256.7, B 1675.3370 5038.75 1008.5, 1260.4, B 1680.4 371 5009.75 1002.8, 1253.1, B1670.3 372 5010.69 1002.9, 1253.4, B 373 4995.68 1000.0, 1249.7, B1665.8 374 5025.71 1006.0, 1257.3, B 1676.2 375 5009.71 1002.9, 1253.2,B 1670.8 376 4995.68 1000.0, 1249.7, B 1665.8 377 5007.73 1002.3,1252.7, A 1669.9 378 5023.73 1005.8, 1256.8, B 1675.9 379 5035.741008.0, 1259.7, B 1679.1 380 5023.73 1256.7 B 381 4979.59 1245.7 B 3824966.64 B 383 4965.7 B B 384 4966.64 B 385 4947.64 990.3, 1237.7, 1649.8B 386 5007.73 1002.3, 1252.8, B 1669.8 387 4952.61 991.5, 1239.0, 1651.4B 388 4951.67 991.2, 1238.7, 1650.9 B 389 5081.77 1017.2, 1271.3, B1694.6 390 5067.74 1014.3, 1267.8, B 1690.0 391 4951.67 991.2, 1238.7,1651.2 B 392 4952.61 991.3, 1238.9, 1651.6 B 393 4952.66 991.3, 1238.9,1651.7 B 394 4952.61 991.4, 1238.9, 1651.7 B 395 4949.59 990.8, 1238.2,1650.6 B 396 4642.31 929.3, 1161.3, 1548.1 B 397 4741.44 1186.1 B 3984912.59 983.3, 1228.9, 1638.1 B 399 4991.67 999.2, 1248.8, 1664.6 B 4005037.76 1008.5, 1260.2, B 1679.8 401 5023.73 1005.5, 1256.7, B 1675.2402 4966.64 994.2, 1242.4, 1656.3 B 403 4947.64 990.3, 1237.7, 1650.1 A404 4965.7 994.0, 1242.2, 1655.9 B 405 4966.64 994.0, 1242.4, 1656.3 B406 4966.64 994.1, 1242.4, 1656.3 A 407 4966.64 994.2, 1242.4, 1656.3 A408 4957.67 992.3, 1240.2, 1653.3 B 409 4957.67 992.3, 1240.2, 1653.2 B410 5023.73 1005.6, 1256.7 B 411 4966.68 994.1, 1242.4, 1656.3 B 4124966.64 994.2, 1242.4, 1656.2 B 413 4091.62 1023.7, 1364.6 B 414 4219.791055.8, 1407.2 A 415 4347.96 870.6, 1087.8, 1450.1 A 416 4477.08 896.3,1120.1, 1493.1 B 417 4624.25 925.7, 1156.8, 1542.1 B 418 4752.42 951.3,1188.8, 1584.8 B 419 4823.5 965.5, 1206.7, 1608.7 B B 420 4880.55 977.0,1220.9, 1627.4 B 421 4952.61 991.3, 1238.9, 1651.4 B 422 5010.65 1002.8,1253.4, B 1670.8 424 4955.71 992.0; 1239.8; 1652.5 C C 425 4718.411180.4; 1573.5 C C 426 4884.82 977.8; 1222.0; 1629.1 B C 427 4658.631165.5; 1553.7 B B 428 4361.04 1091.2, 1454.6 D 429 4491.15 899.0;1123.6; 1497.8 D 430 4393.04 879.5; 1099.1; 1465.2 D 431 4335.99 868.1;1084.8; 1446.1 D 432 4393.04 879.5; 1099.1; 1465.1 D 433 4300.95 861.1;1076.1; 1434.4 D 434 4342.04 869.2; 1086.4; 1448.1 D 435 4359.01 872.8;1090.6; 1453.8 C 436 4375 875.9; 1094.8; 1459.1 C 437 2921.47 731.3;974.6; 1461.3 A 438 3349.95 838.3; 1117.4; 1675.6 A 439 4328.96 1083.1;1443.7 C 440 4017.62 1005.3; 1339.9 B 441 3584.99 897.1; 1195.8; 1793.4B 442 3586.09 897.3; 1196.2; 1793.7 A 443 3657.25 915.2; 1219.8; 1829.3A 444 3586.18 897.4; 1196.1; 1794.1 B 445 4920.66 985.0; 1230.9; 1640.8A 446 4611.3 923.2; 1153.6; 1537.9 B 447 3870.49 968.5; 1290.9; 1936.1 C448 4104.74 1027.0; 1368.9 D 449 4053.61 811.6; 1014.2; 1352.0 B 4504200.83 1051.1; 1401.0 B 451 4271.91 1068.8; 1424.8 B

Example 3

PAC1 peptide antagonists were also evaluated in an in vivopharmacodynamic model—a model of maxadilan-induced increase in bloodflow. Since maxadilan is a highly selective agonist of PAC1 with noactivity at related GPCRs, VPAC1 and VPAC2, PAC1 peptides were evaluatedbased on their prevention of the maxadilan effect.

In Vivo PD Model (MIIBF)

All procedures in this report were conducted in compliance with theAnimal Welfare Act, the Guide for the Care and Use of LaboratoryAnimals, and the Office of Laboratory Animal Welfare.

Naive male Sprague Dawley® rats aged 8-12 weeks at the time of the studywere purchased from Charles River Laboratories. Animals weregroup-housed in non-sterile, ventilated micro-isolator housing in AmgenAssessment and Accreditation of Laboratory Animal Committee(AAALAC)-accredited facility. Animals had ad libitum access to pelletedfeed (Harlan Teklad 2020X, Indianapolis, Ind.) and water (on-sitegenerated reverse osmosis) via automatic watering system.

All PAC1 peptides were tested in a rat maxadilan-induced increase inblood flow (MIIBF) pharmacodynamic (PD) model with a laser Dopplerimaging. A dosing solution of maxadilan (Bachem, H6734.0500) wasprepared fresh daily by diluted maxadilan stock solution (0.5 mg/mL) in1×PBS to the final concentration of 0.5 μg/mL. All tested peptides wereprepared in 1×PBS at different concentrations depend on the doserequired for the experiment and given in a single bolus i.v. or s.c.injection. A laser Doppler imager (LDI-2, Moor Instruments, Ltd,Wilmington, Del.) was used to measure dermal blood flow (DBF) on theshaved skin of the rat abdomen with a low-power laser beam generated bya 633 nm helium-neon bulb. The measurement resolution was 0.2 to 2 mm,with scanning distances between the instrument aperture and the tissuesurface of 30 cm. In this report, DBF was measured and expressed as %change from baseline [100×(individual post-CAP flux-individual baselineflux)/individual baseline flux] and further calculated to % Inhibitionto quantify the magnitude of drug effect [Mean of vehicle % change fromBL−individual drug treated rat % change from BL)/Mean of vehicle %change from BL].

On the test day, following anesthetization with propofol, the rat'sabdominal area was shaved and each animal was placed in a supineposition on a temperature-controlled circulating warm-water pad tomaintain a stable body temperature during the study. After a 10 to 15minute stabilization period, a rubber O-ring (0.925 cm inner diameter,0-Rings West, Seattle, Wash.) was placed on the rat abdomen withoutdirectly positioning it over a visible blood vessel while the rat placedin a supine position on a temperature-controlled circulating warm-waterpad to maintain a stable body temperature during the study. Afterplacement of an O-ring on the selected area, a baseline (BL) DBFmeasurement was taken. After the BL scan, PAC1 peptides wereadministrated either 5 min for i.v., or 15 min for s.c. then followed by20 μL (in 0.5 μg/mL) intradermal injection of maxadilan at the center ofthe O-ring. The post-maxadilan DBF was measured at 30 min and the postpeptides time was 35 min for i.v. and 45 min for s.c. respectively. TheO-ring serves as an area of interest in which the DBF will be analyzedwithin the O-ring.

All DBF results were expressed as the mean±SEM. A one-way ANOVA followedby Dunnett's Multiple Comparison Test (MCT) was used to assess thestatistical significance of PAC1 peptides effects relative to thevehicle within the study. A p<0.05 was used to determine significancebetween any two groups.

Single Bolus Intravenous (i.v.) Dose of PAC1 Peptide Treatment in MIIBF

Pretreatment of peptides 5 min prior to maxadilan challenge (20 μl in0.5 μg/mL) at a dose range from 0.03 to 0.7 mg/kg prevented the MIIBF.At 35 min post peptide treatment, there was a statistically significantinhibition for SEQ ID NOs 283, 253, 250, 249, 233, 190, 180, 179, 183,181 at the lowest dose of 0.1 mg/kg.

Single Bolus Subcutaneous (s.c.) Dose of PAC1 Peptide Treatment in MIIBF

Pretreatment of peptides 15 min prior to maxadilan challenge at a doserange from 0.1 to 2 mg/kg prevented the maxadilan-induced increase inDBF. At 45 min post peptide treatment, there was a statisticallysignificant inhibition sequence SEQ ID #156, 151, 180, 105, 163, 86, 88,85, 77, 94, 152, 56, 58, 65 at the lowest dose of 0.3 mg/kg.

Example 4 Yeast Display Affinity Maturation

To engineer an anti-PAC1 peptide with improved activity, affinitymaturation was performed by constructing yeast-displayed libraries ofcontrol peptide mutants and sorting for improved binding to theextracellular domain (ECD) of human PAC1 using fluorescence-assistedcell sorting (FACS). Three mutant libraries were designed tocomprehensively query mutation combinations in regions of the peptidewhere point mutation previously led to modest potency improvements. Thesurface-exposed residues that would most likely make contacts to the ECDwere targeted for saturation mutagenesis. Additional partially buriedresidues that may influence the presentation of neighboringsurface-exposed sidechains were also selected for limited, conservativemutagenesis. To restrict theoretical diversities of each combinatoriallibrary to a manageable number for yeast display, the chosen residuesfor mutagenesis were grouped by secondary structure element and threeseparate libraries were constructed to explore these regions. Anadditional library was designed to subtly alter the global presentationof surface-exposed sidechains through repacking of the hydrophobic coreof the peptide.

For each of the four designed libraries, oligonucleotides encoding thedesigned amino acid diversities were assembled into full-length peptidegenes using PCR, and the resulting PCR products were co-transformed intoyeast along with a yeast display vector that genetically fused HA andc-myc tags to the N- and C-termini of the peptides, respectively. Thefour constructed yeast-displayed peptide libraries ranged from 1.2E08 to1.7E08 in size and covered the designed theoretical diversities by atleast ten-fold.

Three rounds of binding selection on each yeast-displayed peptidelibrary were performed using FACS. For each round, yeast library poolswere resuspended in phosphate-buffered saline supplemented with 0.5%bovine serum albumin incubated with biotinylated human PAC1 ECD andstained with anti-HA antibody-AlexaFluor647 (for display) andstreptavidin-phycoerthythrin (PE) conjugates (for PAC1 binding) prior tosorting. The AlexaFluor647/PE double-positive cells that exhibitedimproved binding (per unit of display) compared to identically treatedpeptide controls were collected and grown to saturation. To enter thenext round of selection, a ten-fold excess of cells relative to thenumber recovered from the previous round of sorting were induced fordisplay. To increase selection stringency with each successive round,the concentration of biotinylated PAC1 ECD was lowered by at leastfive-fold (e.g. 1 nM to 0.2 nM), a smaller percentage of the top-bindingcells was collected, or a selection strategy to enrich for mutantsexhibiting the slowest binding off-rates was implemented.

After completion of three rounds of sorting for each library, over 800individual yeast colonies from all four libraries were plated and pickedto screen for superior human PAC1 binding compared to theyeast-displayed peptide benchmarks. The peptides were tested for bindingto rat PAC1 ECD and specificity for PAC1 in a second round of flowcytometry binding screens conducted under more stringent conditions. Thesequences of the top mutants, which exhibited the most improved bindingto 0.05 nM human and rat PAC1 ECD and no detectable binding to 5 nM ofan irrelevant protein, were delivered for peptide synthesis as describedin Example 1.

Example 5 In Vitro Assay of Peptide Stability

To model degradation in blood, PAC1 peptides of the present applicationwere incubated in vitro with rat plasma. The stability analysis wasconducted in a manner similar to as previously described by Souverain etal. (J. of Pharmaceutical and Biomedical Analysis, 35 (2004) 913-920).

Briefly, PAC1 peptides were spiked into rat plasma and incubated at 37°C. for up to 6 hours. Employing the reverse stability technique forvarious time points, plasma samples were quenched by the addition ofperchloric acid in water containing internal standard (IS) and vortexedundergoing protein precipitation extraction (PPE). Following the vortexmix, samples were centrifuged and the supernatant containing PAC1peptide and IS was subsequently analyzed using a liquid chromatographymass-spectrometry (LC-MS) system (Sciex® API 5500). The peak area ratioof intact PAC1 peptide to IS was determined for each time point. Peptidestability was reported as a percentage of the peak area ratio at eachtime point relative to the peak are ratio of a baseline sample(pre-incubation).

Representative stability data are presented in Table 4.

TABLE 4 PAC1 Peptide Stability in Rat Plasma (% Intact PeptideRemaining) Time Seq ID# [h] #283 #197 #164 #151 #150 #140 #88 #77 0 100100 100 100 100 100 100 100 0.5 87.5 95.2 94.3 119.9 116.7 100.7 81.497.9 1 112.4 98.9 113.3 87.0 89.7 91.4 90.8 105.6 2 101.2 91.9 107.7103.5 92.1 93.4 84.0 104.1 4 114.6 89.0 106.2 111.5 90.2 106.9 110.189.6 6 98.9 87.5 96.5 107.1 92.8 108.4 100.1 109.9

The majority of peptides evaluated demonstrated good stability for 6hours (>90%).

What is claimed is:
 1. A peptide, or a pharmaceutically acceptable saltthereof, wherein the peptide is of SEQ ID NO:1-SEQ ID NO:3, SEQ IDNO:5-SEQ ID NO:37, SEQ ID NO:39-SEQ ID NO:48, SEQ ID NO:51-SEQ IDNO:164, SEQ ID NO:166-SEQ ID NO:194, SEQ ID NO:197-SEQ ID NO:211, SEQ IDNO:213-SEQ ID NO:271, SEQ ID NO:273-SEQ ID NO:304, SEQ ID NO:306-SEQ IDNO:418, SEQ ID NO:420-SEQ ID NO:422 or SEQ ID NO:424-451.
 2. The peptideaccording to claim 1, or a pharmaceutically acceptable salt thereof,wherein the peptide is SEQ ID NO: 151, 180, 163, 86, 88, 85, 94, 65, 56,152, or
 448. 3. A composition comprising the peptide of claim 1, or apharmaceutically acceptable sale thereof and a pharmaceuticallyacceptable excipient.
 4. A composition comprising the peptide of claim2, or a pharmaceutically acceptable sale thereof and a pharmaceuticallyacceptable excipient.